Methods for Production of Xylitol in Microorganisms

ABSTRACT

The invention provides biosynthetic routes to xylitol production that do not require pure  D -xylose for synthesis and that can utilize inexpensive substrates such as hemicellulose hydrolysates.

PRIORITY

This application is a divisional application of U.S. Ser. No.11/133,045, filed May 19, 2005, now allowed, which claims priority toU.S. Ser. No. 11/133,025, filed May 19, 2005, now abandonded. Thisapplication also claims the benefit of U.S. Provisional Application Ser.No. 60/572,588, filed May 19, 2004; 60/620,173, filed Oct. 18, 2004; and60/572,438, filed May 19, 2004, all of which are incorporated byreference herein in their entirety.

FIELD OF THE INVENTION

The invention is in the field of constructing effective biosyntheticroutes to xylitol production that do not require pure D-xylose forsynthesis and that can utilize inexpensive substrates such ashemicellulose hydrolysates.

BACKGROUND OF THE INVENTION

Xylitol is currently produced by chemical hydrogenation of xylosepurified from xylan hydrolysates. The use of microorganisms to producexylitol and other polyols from inexpensive starting materials such ascorn and other agricultural byproduct and waste streams has long beenthought to be able to significantly reduce production costs for thesepolyols as compared to chemical hydrogenation. Such a process wouldreduce the need for purified xylose, produce purer, easier to separateproduct, and be adaptable to a wide variety of raw materials fromdifferent geographic locations.

Despite a significant amount of work, development of a commerciallyfeasible microbial production process has remained elusive for a numberof reasons. To date, even with the advent of genetically engineeredyeast strains, the volumetric productivity of the strains developed donot reach the levels necessary for a commercially viable process.

Xylitol is currently produced from plant materials—specificallyhemicellulose hydrolysates. Different plant sources contain differentpercentages of cellulose, hemicellulose, and lignin making most of themunsuitable for xylitol production. Because of purity issues, only thehydrolysate from birch trees is used for xylitol production. Birch treehydrolysate is obtained as a byproduct of the paper and pulpingindustry, where lignins and cellulosic components have been removed.Hydrolysis of other xylan-rich materials, such as trees, straws,corncobs, oat hulls under alkaline conditions also yields hemicellulosehydrolysate, however these hydrolysates contain many competingsubstrates. One of these substrates, L-arabinose is a particular problemto xylitol production because it can be converted to L-arabitol, whichis practically impossible to separate from xylitol in a cost effectiveway.

D-xylose in the hydrolysate is converted to xylitol by catalyticreduction. This method utilizes highly specialized and expensiveequipment for the high pressure (up to 50 atm) and temperature (80-140°C.) requirements as well as the use of Raney-Nickel catalyst that canintroduce nickel into the final product. There have been severalprocesses of this type described previously, for example U.S. Pat. Nos.3,784,408, 4,066,711, 4,075,406, 4,008,285, and 3,586,537. In addition,the xylose used for the chemical reduction must be substantiallypurified from lignin and other cellulosic components of thehemicellulose hydrolysate to avoid production of extensive by-productsduring the reaction.

The availability of the purified birch tree hydrolysate startingmaterial severely limits the xylitol industry today. If a specific,efficient reduction process could be developed that could convert xyloseand arabinose to xylitol, but not reduce any other impurities that werepresent in a starting mixture, then a highly cost competitive processcould be developed that would allow significant expansion of the xylitolmarket.

Many of the prior art methods of producing xylitol use purified D-xyloseas a starting material and will also generally convert L-arabinose toL-arabitol (and other sugars to their respective reduced sugar polyol).While there has been a significant amount of work on the development ofan organism to convert D-xylose to xylitol, none of the prior artapproaches have been commercially effective. There are several reasonsfor this. First, D-xylose utilization is often naturally inhibited bythe presence of glucose that is used as a preferred carbon source formany organisms. Second, none of the enzymes involved have been optimizedto the point of being cost effective. Finally, D-xylose in its pure formis expensive. Prior art methods do not address the need for alternativestarting materials. Instead they require relatively pure D-xylose.Agricultural waste streams are considered to be the most cost-effectivesource of xylose. These waste streams are generally mixed with a varietyof other hemicellulosic sugars (L-arabinose, galactose, mannose, andglucose), which all affect xylitol production by the microbes inquestion. See, Walthers et al. (2001). “Model compound studies:influence of aeration and hemicellulosic sugars on xylitol production byCandida tropicalis.” Appl Biochem Biotechnol 91-93:423-35. However, ifan organism can be engineered to utilize more than one of the sugars inthe waste stream, it would make the process much more cost effective.

In addition to xylose, L-arabinose is an abundant sugar found inhemicellulose ranging from 5% to 20% depending on the source.Co-conversion of L-arabinose to xylitol or cell biomass would allow agreater variety of starting materials to be used (birch has very lowarabinose content and thus does not lead to production of L-arabitolduring the chemical hydrogenation). Therefore, methods of convertingxylose and arabinose to xylitol, converting xylose and arabinose toxylitol while the arabinose remains unconverted, and converting xyloseto xylitol and arabinose to biomass would be desirable.

A variety of approaches have been reported in the literature for thebiological production of xylitol. While some basic research has beenperformed, development of an effective bioprocess for the production ofxylitol has been elusive. Many of the systems described below sufferfrom problems such as poor strain performance, low volumetricproductivity, and too broad of a substrate range. Of these, yeasts,primarily Candida, have been shown to be the best producers of xylitolfrom pure D-xylose. See, Hahn-Hagerdal, et al., Biochemistry andphysiology of xylose fermentation by yeasts. Enzyme Microb. Technol.,1994. 16:933-943; Jeffries & Kurtzman, Strain selection, taxonomy, andgenetics of xylose-fermenting yeasts. Enzyme Microb. Technol., 1994.16:922-932; Kern, et al., Induction of aldose reductase and xylitoldehydrogenase activities in Candida tenuis CBS 4435. FEMS MicrobiolLett, 1997. 149(1):31-7; Saha & Bothast, Production of xylitol byCandida peltata. J Ind Microbiol Biotechnol, 1999. 22(6):633-636; Saha &Bothast, Microbial production of xylitol, in Fuels and Chemicals fromBiomass, Saha, Editor. 1997, American Chemical Society. p. 307-319.These include Candida strains C. guilliermondii, C. tropicalis, C.peltata, C. milleri, C. shehatae, C. boidinii, and C. parapsilosis. C.guillermondii is one of the most studied organisms and has been shown tohave a yield of up-to 75% (g/g) xylitol from a 300 g/l fermentationmixture of xylose. See, Saha & Bothast, Production of xylitol by Candidapeltata. J Ind Microbiol Biotechnol, 1999. 22(6):633-636. C. tropicalishas also been shown to be a relatively high producer with a cellrecycling system producing an 82% yield with a volumetric productivityof 5 g L⁻¹ h⁻¹ and a substrate concentration of 750 g/l. All of thesestudies however, were carried out using purified D-xylose as substrate.

Bolak Co., Ltd, of Korea describes a two-substrate fermentation with C.tropicalis ATCC 13803 using glucose for cell growth and xylose forxylitol production. The optimized fed-batch fermentation resulted in 187g L⁻¹ xylitol concentration, 75% g/g xylitol/xylose yield and 3.9 gxylitol L⁻¹ H⁻¹ volumetric productivity. See, Kim et al., Optimizationof fed-batch fermentation for xylitol production by Candida tropicalis.J Ind Microbiol Biotechnol, 2002. 29(1):16-9. The range of xyloseconcentrations in the medium ranged from 100 to 200 g L⁻¹ total xyloseplus xylitol concentration for maximum xylitol production rate andxylitol yield. Increasing the concentrations of xylose and xylitolbeyond this decreased the rate and yield of xylitol production and thespecific cell growth rate, and the authors speculate that this wasprobably due to the increase in osmotic stress. Bolak disclosed thisapproach to xylitol production. See e.g., U.S. Pat. No. 5,998,181; U.S.Pat. No. 5,686,277. They describe a method of production using a novelstrain of Candida tropicalis KCCM 10122 with a volumetric productivityin 3 to 5 L reactions ranging from 3.0 to 7.0 g xylitol L⁻¹ H⁻¹,depending on reaction conditions. They also describe a strain, Candidaparapsilosis DCCM-10088, which can transform xylose to xylitol with amaximum volumetric productivity of 4.7 g xylitol L⁻¹ H⁻¹, again in benchscale fermentation ranging from 3 to 5 liters in size. While C.tropicalis has had moderate success in achieving relatively large levelsof xylitol production than the other strains, it suffers from the factthat it is an opportunistic pathogen, and therefore is not suitable forfood production and the enzyme also makes L-arabitol from L-arabinose.

One promising approach that has only been moderately explored is thecreation of recombinant strains capable of producing xylitol. Xyrofinhas disclosed a method involving the cloning of a xylose reductase genefrom certain yeasts and transferring the gene into a Saccharomycescerevisiae. See, U.S. Pat. No. 5,866,382. The resulting recombinantyeast is capable of reducing xylose to xylitol both in vivo and invitro. An isolated enzyme system combining xylitol reductase withformate dehydrogenase to recycle the NADH cofactor during the reactionhas been described. In this instance, the enzymatic synthesis of xylitolfrom xylose was carried out in a fed-batch bioreactor to produce 2.8 g/lxylitol over a 20 hour period yielding a volumetric productivity ofabout 0.4 g l⁻¹ H⁻¹. See, Neuhauser et al., A pH-controlled fed-batchprocess can overcome inhibition by formate in NADH-dependent enzymaticreductions using formate dehydrogenase-catalyzed coenzyme regeneration.Biotechnol Bioeng, 1998. 60(3):277-82. The use of this on a large scaleusing crude substrate has yet to be demonstrated and poses severaltechnical hurdles.

Several methods for producing xylitol from xylose-rich lignocellulosichydrolyzates through fermentative processes have been described. Xyrofindiscloses a method for the production of substantially pure xylitol froman aqueous xylose solution. See, U.S. Pat. No. 5,081,026; U.S. Pat. No.5,998,607. This solution may also contain hexoses such as glucose. Theprocess uses a yeast strain to convert free xylose to xylitol while thefree hexoses are converted to ethanol. The yeast cells are removed fromthe fermentation by filtration, centrifugation or other suitablemethods, and ethanol is removed by evaporation or distillation.Chromatographic separation is used to for final purification. Theprocess is not commercially viable because it requires low arabinosewood hydrolyzate to prevent L-arabitol formation and the total yield was(95 g l⁻¹) and volumetric productivity is low (1.5 g l⁻¹ H⁻¹). Xyrofinalso discloses a method for xylitol synthesis using a recombinant yeast(Zygosaccharomyces rouxii) to convert D-arabitol to xylitol. See, U.S.Pat. No. 5,631,150. The recombinant yeast contained genes encodingD-arbinitol dehydrogenase (E.C. 1.1.1.11) and xylitol dehydrogenase(E.C. 1.1.1.9), making them capable of producing xylitol when grown oncarbon sources other than D-xylulose or D-xylose. The total yield (15 gl⁻¹) and volumetric productivity (0.175 g l⁻¹ H⁻¹) coupled with the useof D-arabitol as starting material make this route highly unlikely tosucceed. Additionally, a 2-step fermentation of glucose to D-arabitolfollowed by fermentation of D-arabitol to xylitol has also beendescribed. See, U.S. Pat. No. 5,631,150; U.S. Pat. No. 6,303,353; U.S.Pat. No. 6,340,582. However, a two-step fermentation is not economicallyfeasible.

Another method of making xylitol using yeasts with modified xylitolmetabolism has been described. See, U.S. Pat. No. 6,271,007. The yeastis capable of reducing xylose and using xylose as the sole carbonsource. The yeast have been genetically modified to be incapable ordeficient in their expression of xylitol dehydrogenase and/or xylulosekinase activity, resulting in an accumulation of xylitol in the medium.A major problem with this method is that a major proportion of theD-xylose is consumed for growth rather than being converted to thedesired product, xylitol.

A process describing the production of xylitol from D-xylulose byimmobilized and washed cells of Mycobacterium smegmatis has beendescribed. See, Izumori & Tuzaki, Production of Xylitol from D-Xylulosebr Mycobacterium smegmatis. J. Ferm. Tech., 1988. 66(1):33-36. Modesttiters of ˜15 g l⁻¹ H⁻¹ were obtained with a 70% conversion efficiencyof D-xylulose into xylitol. Also disclosed was the conversion ofD-xylose into xylitol by using a combination of commercially available,immobilized xylose isomerase and M. smegmatis cells containing xylitoldehydrogenase activity. It was found that xylitol inhibition of thexylose isomerase caused the incomplete conversion of D-xylose intoxylitol. This process does not teach how one could relieve theinhibition of the xylose isomerase by xylitol or how one would engineera single strain to convert D-xylose into xylitol.

Ajinomoto has several patents/patent applications concerning thebiological production of xylitol. In U.S. Pat. No. 6,340,582, they claima method for producing xylitol with a microorganism containingD-arbinitol dehydrogenase activity and D-xylulose dehydrogenaseactivity. This allows the organisms to convert D-arbinitol to D-xyluloseand the D-xylulose to xylitol, with an added carbon source for growth.Sugiyama further develops this method in U.S. Pat. No. 6,303,353 with alist of specific species and genera that are capable of performing thistransforming, including Gluconobacter and Acetobacter species. This workis furthered by the disclosure of the purified and isolated genes fortwo kinds of xylitol dehydrogenase from Gluconobacter oxydans and theDNA and amino acid sequences, for use in producing xylitol fromD-xylulose. See, U.S. Pat. Publ. 2001/0034049; U.S. Pat. No. 6,242,228.In US Appl. Publ. No. 2003/0148482 they further claim a microorganismengineered to contain a xylitol dehydrogenase, that has an ability tosupply reducing power with D-xylulose to produce xylitol, particularlyin a microorganism that has an ability to convert D-arbinitol intoD-xylulose.

Ajinomoto has also described methods of producing xylitol from glucose.Takeuchi et al. in U.S. Pat. No. 6,221,634 describes a method forproducing either xylitol or D-xylulose from Gluconobacter, Acetobacteror Frateuria species from glucose. However, yields of xylitol were lessthan 1%. Mihara et al. further claim specific osmotic stress resistantGluconobacter and Acetobacter strains for the production of xylitol andxylulose from the fermentation of glucose. See, U.S. Pat. No. 6,335,177.They report a 3% yield from a 20% glucose fermentation broth. In U.S.Pat. Appl. No. 2002/0061561, Mihara et al. claim further discoveredstrains, also with yields of only a few percent. See, U.S. Pat. No.6,335,177.

Cerestar has disclosed a process of producing xylitol from a hexose suchas glucose in two steps. See, U.S. Pat. No. 6,458,570. The first step isthe fermentative conversion of a hexose to a pentitol, for example,glucose to arabitol, and the second step is the catalytic chemicalisomerisation of the pentitol to xylitol.

Bley et al. disclose a method for the biotechnological production ofxylitol using microorganisms that can metabolize xylose to xylitol. See,WO03/097848. The method comprises the following steps: a) microorganismsare modified such that oxidation of NADH by enzymes other than thexylose reductase is reduced or excluded; b) the microorganisms arecultivated in a substrate containing xylose and 10-40 grams per liter ofsulphite salt (e.g. calcium hydrogen sulphite, natrium sulphite,potassium sulphite); c) the microorganisms are cultivated in an aerobicgrowth phase and an oxygen-limited xylitol production phase; and d) thexylitol is enriched and recovered from the substrate.

Londesborough et al. have disclosed a genetically modified funguscontaining L-arabitol 4-dehydrogenase and L-xylulose xylulose reductase.See, U.S. Pat. Appl. Publ. No. 2003/0186402. This application is aimedat producing useful products from biomass containing L-arabinose, whichis a major constituent of plant material but does not disclose the useof D-xylose/L-arabinose mixtures for the synthesis of xylitol inprocaryotes. Verho et al. also describe and alternative L-xylulosereductase from Ambrosiozyma monospora that utilizes NADH as co-factor.See, Verho et al., New Enzyme for an in vivo and in vitro Utililizationof Carbohydrates. 2004, Valtion Teknillinen Tutki-muskeskus. p. 15.

Researchers at Danisco have developed several xylitol bioprocesses.Heikkila et al. describes a process wherein purified L-xylose isutilized as intermediate. See, U.S. Pat. Appl. Publ. No. 2003/0097029.The application also covers methods of production of L-xylose. Thisprocess is not feasible because L-xylose is a rare sugar and isconsiderably more valuable than the final product. A method forsimultaneously producing xylitol as a co-product during fermentativeethanol production, utilizing hydrolyzed lignocellulose-containingmaterial is disclosed in U.S. Pat. Appl. Publ. No. 2003/0235881. Thisprocess consists of fermenting the free hexoses to ethanol while thexylose is converted to xylitol with a single yeast strain. The yields,however, of both ethanol and xylitol were relatively poor and requirepure D-xylose as a substrate. Danisco has also developed a multipleprocesses for the preparation of xylitol, all of them utilizingribulose. See, U.S. Pat. Appl. Publ. No. 2003/0125588. These processesinclude different conversion reactions, such as reduction, epimerizationand/or isomerisation. Xylitol is also produced in the fermentation ofglucose in one embodiment. The process can also use ribulose andxylulose as starting material, followed by reduction, epimerization andisomerisation to xylitol. Again the starting substrates D-xylulose andribulose are more valuable than the final product.

Ojamo et al. shows a method for the production of xylitol involving apair of microorganisms one having xylanolytic activity, and anothercapable of converting a pentose sugar to xylitol, or a singlemicroorganism capable of both reactions. See, U.S. Pat. Appl. Publ. No.2004/0014185. In one embodiment of the invention, two microorganisms areused for the production of xylitol, one microorganism possessingxylanolytic activity and the other possessing the enzymatic activityneeded for conversion of a pentose sugar, such as D-xylose andL-arabinose, preferably D-xylose, to xylitol. This method requires acomplicated two-organism system and produces mixtures of xylitol andL-arabitol, which need extra purification and recycle steps to improvethe xylitol yield. It does not teach simple, single organism methodsthat can use D-xylose/L-arabinose mixtures to synthesize pure xylitol.Finally, Miasnikov et al. have developed multiple methods for theproduction of xylitol, five-carbon aldo- and keto-sugars and sugaralcohols by fermentation in recombinant hosts. See, U.S. Pat. Appl.Publ. No. 2003/0068791. These recombinant hosts have been engineered toredirect pentose phosphate pathway intermediates via ribulose-5-P,xylulose-5-P and xylitol-5-P into the production of xylitol,D-arbinitol, D-arabinose, D-xylose, ribitol, D-ribose, D-ribulose,D-xylose, and/or D-xylulose. Methods of manufacturing are disclosed thatuse such hosts, but the productivity is low.

While clearly there has been a significant amount of work on thedevelopment of an organism to convert xylose to xylitol, none of thesehave resulted in an effective production organism or a commercializedprocess. The yeast methods described above all require relatively purexylose as a starting material, since the organisms described will alsoconvert L-arabinose to L-arabitol (and other sugars to their respectivereduced sugar pentitol). This results in difficult-to-remove by-productswhich can only be separated by costly separation methods. Purifiedxylose is also prohibitively expensive for use in a bioprocess andcannot compete with the current chemical hydrogenation. Several of theprocesses above consist of more than one fermentation step, which isagain, cost-prohibitive. The reported production rate of some of thestrains is low, as in the Ajinomoto patents. Above all, none of theenzymes or strains involved has been engineered to be cost effective. Ifthe turnover rate of one or more enzyme can be improved, then theproduction level would increase. Further, none of the approaches haveaddressed the problems associated with the use of agriculturalhydrolyzates to produce xylitol. Agricultural waste streams areconsidered to be the most cost-effective source of D-xylose. These wastestreams are generally mixed with a variety of other hemicellulosicsugars (arabinose, galactose, mannose, and glucose), which all affectxylitol production by the microbes in question. See, Walthers et al.,Model compound studies: influence of aeration and hemicellulosic sugarson xylitol production by Candida tropicalis. Appl Biochem Biotechnol,2001. 91-93:423-35.

Hence there is an opportunity for a high-specificity bioprocess that isboth economical and safe and can utilize alternative starting materials.Table 1 outlines several potential agricultural residues that would besuitable as feedstocks if such a process was available. The instantinvention addresses these problems and allows the engineering of anefficient bioprocess for making xylitol.

SUMMARY OF THE INVENTION

One embodiment of the invention provides a recombinant bacterium thatexpresses proteins comprising xylose reductase, L-arabitol dehydrogenaseor ribitol dehydrogenase, or both, and L-xylulose reductase activities,wherein the recombinant bacterium can produce an end-product of xylitolfrom substrates comprising: D-xylose; or L-arabinose; or L-arabinose andD-xylose; or L-arabinose, D-xylose and other sugars; and wherein nosubstantial amount of L-arabitol is produced as an end-product. Thesubstrate can be a xylan hydrolysate or a hemicellulose hydrolysate. Therecombinant bacterium can further express a ribitol transporter protein.The bacterium can be Escherichia coli. The recombinant bacterium can benon-pathogenic. The bacterium can have an inactive ptsG gene or amissing ptsG gene. The recombinant bacterium can produce L-arabitol orL-xylulose or both as intermediates to the xylitol end-product. Therecombinant bacterium can comprise one or more recombinant nucleic acidsequences encoding aldose reductase, L-xylose reductase, ribitoldehydrogenase, ribitol transporter protein, L-arabitol dehydrogenase,and L-xylulose reductase. The nucleic acid sequence encoding xylosereductase can be a Pichia stipitis nucleic acid sequence or a yafB oryajO nucleic acid sequence from E. coli. The nucleic acid sequenceencoding ribitol dehydrogenase can be a Klebsiella pneumoniae orKlebsiella aerogenes nucleic acid sequence. The nucleic acid sequenceencoding L-xylulose reductase can be an Ambrosioyma monospora nucleicacid sequence. The nucleic acid sequence encoding L-arabitoldehydrogenase can be a Trichoderma reesei nucleic acid sequence. Thenucleic acid sequence encoding L-xylose reductase can be a T. reeseinucleic acid sequence.

Another embodiment of the invention provides a method for producing axylitol end-product comprising fermenting a substrate comprisingD-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose,D-xylose and other sugars with a recombinant bacterium comprising xylosereductase, L-arabitol dehydrogenase, and L-xylulose reductaseactivities, wherein the recombinant bacterium can produce an end-productof xylitol from substrates comprising: D-xylose; or L-arabinose; orL-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars; andwherein no substantial amount of L-arabitol is produced as anend-product. L-arabitol, or L-xylulose or both can be produced as anintermediate to the xylitol end-product. The method does not requireseparation of L-arabitol from the xylitol end-product.

Yet another embodiment of the invention provides a method for producingL-xylulose comprising fermenting a substrate comprising D-xylose; orL-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose andother sugars with a recombinant bacterium comprising xylose reductase,L-arabitol dehydrogenase, and L-xylulose reductase activities, whereinthe recombinant bacterium can produce an end-product of xylitol fromsubstrates comprising: D-xylose; or L-arabinose; or L-arabinose andD-xylose; or L-arabinose, D-xylose and other sugars; and wherein nosubstantial amount of L-arabitol is produced as an end-product.L-xylulose is collected before it is converted to xylitol.

Still another embodiment of the invention provides a method of producingxylitol from a substrate comprising D-xylose; or L-arabinose; orL-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars. Themethod comprises contacting the substrate with one or more isolatedbacteria that comprise xylose reductase activity, L-arabitoldehydrogenase activity or ribitol dehydrogenase activity or both, andL-xylulose reductase activity, wherein the substrate is converted to anend-product of xylitol and wherein substantially no L-arabitol isproduced as an end-product. The one or more bacteria can also compriseribitol transporter activity.

Even another embodiment of the invention provides a process forproducing xylitol from a substrate comprising D-xylose; or L-arabinose;or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars.The method comprises contacting the substrate with xylose reductase,L-arabitol dehydrogenase or ribitol dehydrogenase or both, andL-xylulose reductase, wherein the substrate is converted to anend-product of xylitol and wherein substantially no L-arabitol isproduced as an end-product. L-arabitol or L-xylulose or both can beproduced as intermediate products. The substrate can be furthercontacted with ribitol transporter protein.

Yet another embodiment of the invention provides an isolatedmicroorganism comprising xylose specific reductase activity, wherein thexylose specific reductase activity does not convert L-arabinose toL-arabitol. The microorganism can produce an end-product of xylitol froma substrate comprising D-xylose; or L-arabinose; or L-arabinose andD-xylose; or L-arabinose, D-xylose and other sugars. The microorganismcan produce no substantial amount of L-arabitol as an end-product. Thesubstrate can be a xylan hydrolysate or hemicellulose hydrolysate. Themicroorganism can be E. coli. The microorganism can have an inactiveptsG gene or a missing ptsG gene. The microorganism can benon-pathogenic. The microorganism can be a bacteria, fungus or yeast.The xylose specific reductase can be encoded by a nucleic acidcomprising SEQ ID NO:43.

Another embodiment of the invention provides a method for producingxylitol comprising fermenting a substrate comprising D-xylose; orL-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose andother sugars; with an isolated microorganism comprising xylose specificreductase activity, wherein the xylose specific reductase activity doesnot convert L-arabinose to L-arabitol. The method does not requireseparation of L-arabitol from xylitol.

Yet another embodiment of the invention provides a purified xylosespecific reductase comprising SEQ ID NO:43. Another embodiment of theinvention provides a purified P. stipitis xylose reductase comprising aSer233Pro mutation and a Phe286Leu mutation.

Still another embodiment of the invention provides a process forproducing xylitol comprising contacting a substrate comprising D-xylose;or L-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xyloseand other sugars; with a xylose-specific reductase. The xylose-specificreductase can comprise SEQ ID NO:43.

Even another embodiment of the invention provides a recombinant E. colithat comprises a nucleic acid sequence encoding xylitol dehydrogenase,wherein the E. coli produces substantially no xylose isomerase. The E.coli can have an inactive or missing PtsG gene.

Still another embodiment of the invention provides a method of screeningfor xylose reductase activity. The method comprises transforming arecombinant E. coli that comprises a nucleic acid sequence encodingxylitol dehydrogenase, and which produces substantially no xyloseisomerase with a nucleic acid molecule encoding a putative xylosereductase to produce a transformant; and adding the transformant toD-xylose minimal media, wherein, if the transformant comprises anexpressed nucleic acid encoding a xylose reductase the transformant willgrow in the D-xylose minimal media.

Another embodiment of the invention provides a recombinant E. colicomprising L-xylulose kinase activity, L-xylulose 5-phosphate epimeraseactivity, and L-ribulose 5-phosphate 4-epimerase activity. The E. colistrain can be strain K12. Another embodiment of the invention provides arecombinant E. coli strain comprising a deleted or inactive yiaJ gene.The strain can be E. coli K12.

Even another embodiment of the invention provides a method of screeningfor L-arabitol dehydrogenase activity or ribitol dehydrogenase activity.The method comprises transforming a recombinant E. coli comprisingL-xylulose kinase activity, L-xylulose 5-phosphate epimerase activity,and L-ribulose 5-phosphate 4-epimerase activity or a recombinant E. colistrain comprising a deleted or inactive yiaJ gene with a nucleic acidmolecule encoding a putative L-arabinitol dehydrogenase or ribitoldehydrogenase to produce a transformant, and adding the transformant toL-arabinitol media, wherein if the transformant comprises an expressednucleic acid encoding a L-arabinitol dehydrogenase, the transformantwill grow in the L-arabinitol media.

Still another embodiment of the invention provides an isolated xylosereductase that is active at 37° C. The xylose reductase can retain 90%or more of its activity at 37° C. when compared to its activity at 30°C. The xylose reductase can comprise an amino acid sequence of SEQ IDNO:44. The xylose reductase can comprise a C. tenuis xylose reductasethat comprises a Gly32Ser mutation and an Asn138Asp mutation.

Yet another embodiment of the invention provides a method for screeningfor bacteria that cannot utilize L-arabinose. The method comprisestransforming bacteria that do not have xylose isomerase activity or anaraBAD operon and that have xylitol dehydrogenase activity andL-ribulokinase activity, with a nucleic acid encoding a xylosereductase, wherein if the xylose reductase is a xylose-specificreductase the transformed bacteria cannot utilize L-arabinose and willgrow on media comprising L-arabinose and D-xylose, and wherein if thexylose reductase is not a xylose-specific reductase, the transformedbacteria can utilize L-arabinose and will not grow on media comprisingL-arabinose and D-xylose.

Even another embodiment of the invention provides an isolatedmicroorganism comprising a recombinant operon comprising a nucleic acidencoding a xylitol dehydrogenase and a nucleic acid encoding a xyloseisomerase.

Another embodiment of the invention provides a method of convertingD-xylose to xylitol comprising fermenting a substrate comprisingD-xylose with an isolated microorganism comprising a recombinant operoncomprising a nucleic acid encoding a xylitol dehydrogenase and a nucleicacid encoding a xylose isomerase. D-xylulose can be produced as anintermediate to the xylitol. Greater than 50% of the D-xylose can beconverted to xylitol. The microorganism can be a bacterium, such as E.coli, a fungus, or a yeast.

Still another embodiment is the selection of a xylose isomerase that isresistant to xylitol and shows enhanced xylitol synthesis when combinedin a strain carrying a xylitol dehydrogenase that can not utilizeD-xylose. Further, this can be optimally by using a bacterial, fungal oryeast host that has been relived of glucose repression so as all of thesugars in hemicellulose hydrolysate can be utilized during xylitolsynthesis.

Another embodiment of the invention provides a purified E. coli xyloseisomerase (xylA) wherein the amino acid sequence comprises the followingmutations:

-   -   (a) F9L, L213Q, F283Y, K311R, H420N;    -   (b) F9L, Q11K, L213Q, F283Y, K311R, H420N;    -   (c) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N; or    -   (d) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N, H439Q.

Even another embodiment of the invention provides a recombinantmicroorganism comprising a mutated E. coli xylose isomerase codingsequence, wherein the microorganism can grow in the presence of about 1%or more of xylitol.

Therefore, the invention provides compositions and methods forconverting xylose and arabinose to xylitol, converting xylose to xylitolwhile any arabinose present remains unconverted, and converting xyloseto xylitol and arabinose to biomass.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a pathway for xylitol synthesis from, for example,hemicellulose.

FIG. 2 shows the properties of a xylose reductase screening strain.

FIG. 3 shows the properties of a L-Arabitol 4-dehydrogenase screeningstrain.

FIG. 4 shows the properties of a D-xylose-specific reductase screeningstrain.

FIG. 5 shows a pathway for production of pure xylitol usinghemicellulose substrate.

FIG. 6 shows a pathway for the production of xylitol from D-xylose.

FIG. 7 shows P. stipitis xylose reductase cloned into vector pTTQ18.

FIG. 8 shows E. coli yafB (xylose reductase) cloned into vector pTTQ18.

FIG. 9 shows Candida tenuis XR gene cloned into pTTQ18.

FIG. 10 shows Trichoderma reesei L-arabitol 4-dehydrogenase gene clonedinto pTTQ18.

FIG. 11 shows Trichoderma reesei L-xylulose reductase gene cloned intopTTQ18.

FIG. 12 shows Trichoderma reesei xylitol dehydrogenase gene cloned intopTTQ18.

FIG. 13 shows construction of the L-arabitol 4-dehydrogenase/L-xylulosereductase operon.

FIG. 14 shows construction of the XR/LAD1 operon.

FIG. 15 shows construction of the yafB/LAD1 operon.

FIG. 16 shows construction of an XR/LAD1/LXR operon.

FIG. 17 shows construction of the yafB/LAD1/LXR operon.

FIG. 18 shows Gluconobacter oxydans xylitol dehydrogenase (xdh) genecloned into pTTQ18.

FIG. 19 shows construction of a constitutive L-xylulose degradationpathway in expression vector pTrp338.

FIG. 20 shows A. monospora L-xylulose reductase cloned into vectorpTTQ18.

FIG. 21 shows Lad1/Alx1 operon cloned into vector pTTQ18.

FIG. 22 shows XR/Lad1/Alx1 operon cloned into vector pTTQ18.

FIG. 23 shows K. pneumoniae ribitol dehydrogenase cloned into vectorpTTQ18.

FIG. 24 shows RbtD/RbtT operon cloned into vector pTTQ18.

FIG. 25 shows RbtD/RbtT/Alx1 operon cloned into vector pTTQ18.

FIG. 26 shows construction of the xylitol dehydrogenase/L-ribulokinase(xdh/araB) operon.

FIG. 27 shows construction of xylitol dehydrogenase/xylose isomerase(xdh/xylA) operon plasmids pZUC35 and pZUC36.

FIG. 28 shows SEQ ID NO:43.

FIG. 29 shows SEQ ID NO:44.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the development of processes, includingwhole-cell microbial processes, using enzyme systems capable ofconverting the following:

-   -   1) Substrates comprising D-xylose and/or L-arabinose (present in        many xylan hydrolysates) mixtures to xylitol;    -   2) Substrates comprising D-xylose and L-arabinose mixtures        wherein the L-arabinose is not converted to L-arabitol.        Xylitol Synthesis from L-Arabinose and D-Xylose

One embodiment of the invention provides a pathway that will producexylitol from substrate sources comprising both L-arabinose and D-xylose.An example of a pathway for this co-conversion is outlined in FIG. 1.One or more xylose reductases (XR) convert L-arabinose and D-xylose toL-arabitol and xylitol, respectively. One or more L-arabitoldehydrogenases (LAD) or ribitol dehydrogenase (RbtD) convert L-arabitolto L-xylulose. One or more xylulose dehydrogenases (LXR) convertL-xylulose to an end-product of xylitol. Therefore, substantially noL-arabitol is present as an end-product. While some researchers havedescribed a 2-step fermentation of glucose to D-arabitol (not L- asdescribed in the instant approach) followed by fermentation ofD-arabitol to xylitol, this two-step process is inherently expensive.See, U.S. Pat. No. 5,631,150; U.S. Pat. No. 6,303,353.

No significant studies at generating a single high-efficiency engineeredmicrobial strain or process for the co-conversion of D-xylose andL-arabinose have been carried out prior to the instant invention.

In one embodiment of the invention the co-conversion of D-xylose andL-arabinose to xylitol occurs by a single recombinant or isolatedmicroorganism. A microorganism can be a bacterium, yeast or fungi. Inone embodiment of the invention the host is an E. coli strain, such asstrain K12. In another embodiment of the invention the microorganismcomprises a deleted or inactive PtsG gene. Deleted means that the codingsequence for PtsG is eliminated from the microorganism. Inactive meansthat the activity of the protein encoded by the gene has less than about25%, 10%, 5%, or 1% of the wild-type protein. Alternatively, inactivemeans that the expression of the gene is reduced by about 75%, 90%, 95%,99% or more as compared to the wild-type gene. In another embodiment ofthe invention two or more recombinant microorganisms can be used in theco-conversion of D-xylose and L-arabinose to xylitol. Each of themicroorganisms can be capable of converting L-arabinose to L-arabitoland D-xylose to xylitol, L-arabitol to L-xylulose, and L-xylulose toxylitol. Alternatively, one or more microorganisms can perform one ormore steps of this pathway, while one or more other microorganisms canperform one or more steps of the pathway wherein an end-product ofxylitol is produced. Optionally, a mixture of microorganisms that canperform one or more steps of the pathway are used.

Substrates of the invention can comprise D-xylose; or L-arabinose; orL-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars.Examples of substrates include xylan hydrolysate and hemicellulosehydrolysate. Agricultural residues that can be used include, forexample, bagasse agricultural residue, corn cob agriculture residue,flax straw agricultural residue, wheat straw residue, oat hullagricultural residue, tree hydrolysate, or a combination thereof.

In one embodiment of the invention a recombinant microorganism processesone or more xylose reductase, L-arabitol dehydrogenase, ribitoldehydrogenase, ribitol transporter, and L-xylulose reductase activities.These activities can be naturally present in the microorganism (i.e.,wild-type) or can be recombinant activities (i.e., a heterologousnucleic acid sequence is added to the microorganism and is expressed bythe microorganism). The recombinant microorganism can comprise one ormore recombinant nucleic acid sequences encoding, for example, xylosereductase, L-arabitol dehydrogenase, ribitol dehydrogenase, ribitoltransporter, and L-xylulose reductase. Methods of making recombinantmicroorganisms are well known in the art. See e.g., Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York (1989), Current Protocols in Molecular Biology, Ausebel et al.(eds), John Wiley and Sons, Inc. New York (2000). Furthermore, methodsof constructing recombinant microorganisms are described in the Examplesbelow.

Xylose reductases generally have broad substrate specificities andfunction on both D-xylose as well as L-arabinose. See, Hahn-Hagerdal etal. (1994). “Biochemistry and physiology of xylose fermentation byyeasts.” Enzyme Microb. Technol. 16: 933-943; Richard et al. (2003).“Production of ethanol from L-arabinose by Saccharomyces cerevisiaecontaining a fungal L-arabinose pathway.” FEM Yeast Res 3(2):185-9.

Many sources of xylose reductases are suitable for use. In oneembodiment of the invention, a xylose reductase of Pichia stipitis isused because its DNA sequence is available, it can use both NADH andNADPH as enzyme cofactor and has good activity on both L-arabinose andD-xylose. Two putative xylose reductases from E. coli (yafB and yajO)could also used due to the ease with which they can be cloned andexpressed in E. coli. XYL1 from Candida tenuis can also be used.

Any L-arabitol dehydrogenase or ribitol dehydrogenase (optionally incombination with ribitol transporter) active in a host of the inventionto convert L-arabitol to L-xylulose can be used. For example, a lad1nucleic acid sequence (L-arabitol 4-dehydrogenase) of Trichoderma reesei(an asexual clonal derivative of Hypocrea jecorina) can be used. See,Richard et al. (2001). “Cloning and expression of a fungal L-arabinitol4-dehydrogenase gene.” J Biol Chem 276(44): 40631-7. L-arabitoldehydrogenases have also been described in Klebsiella pneumoniae andErwinea sp. See, Doten & Mortlock (1984). “Directed evolution of asecond xylitol catabolic pathway in Klebsiella pneumoniae.” J Bacteriol159(2): 730-5; Doten & Mortlock (1985). “Characterization ofxylitol-utilizing mutants of Erwinia uredovora.” J Bacteriol 161(2):529-33; Doten & Mortlock (1985). “Inducible xylitol dehydrogenases inenteric bacteria.” J Bacteriol 162(2):845-8. Additionally, ribitoldehydrogenase (optionally in combination with ribitol transporter) frome.g., K. pneumoniae or K. aerogenes can be used.

Any L-xylulose reductase active in a host of the invention to convertL-xylulose to xylitol can be used. For example, a lxr1 nucleic acidsequence (L-xylulose reductase) from e.g., T. reesei or fromAmbrosiozyma monospora can be used. See, Richard et al. (2002). “Themissing link in the fungal L-arabinose catabolic pathway, identificationof the L-xylulose reductase gene.” Biochemistry 41(20): 6432-7.

Recombinant nucleic acid sequences encoding xylose reductase, L-arabitoldehydrogenase, ribitol dehydrogenase, ribitol transporter, and/orL-xylulose reductase can be either inserted into the chromosome of thehost microorganism or be extra-chromosomal under control of either aconstitutive or inducible promoter. It would also be advantageous toderegulate specific sugar transport systems thus allowing thesimultaneous transport sugars such as D-xylose and L-arabinose whileusing D-glucose as carbon and energy source. The enzymes can also beenhanced by directed evolution to create a host strain that could beused to create unique, commercially viable processes that will useagriculture waste streams to create a valuable product in a costeffective manner.

Xylitol is the desired end-product of the conversions of the invention.An end-product of a conversion reaction can be defined as a desiredproduct that can accumulate in the growth medium of a producing cultureor that can accumulate during a process with a minimal level ofcatabolism and that can be subsequently recovered.

From the starting substrate materials a recombinant or isolatedmicroorganism of the invention can produce L-arabitol and L-xylulose asintermediate products. An intermediate product can be defined as aproduct generated from a starting substrate that requires furtherconversion into an end-product or that can be collected, processed, orremoved separately from an end-product. The intermediate products can becollected before their ultimate conversion to xylitol if desired.

The invention provides methods for producing a xylitol end-productcomprising fermenting a substrate comprising D-xylose; or L-arabinose;or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugarswith one or more recombinant or isolated microorganisms of theinvention. L-arabitol and L-xylulose can be produced as an intermediateto the xylitol end-product. In one embodiment of the invention, themethods do not require separation of L-arabitol from the xylitolend-product because substantially no L-arabitol is produced as anend-product.

One embodiment of the invention provides a process for convertingD-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose,D-xylose and other sugars to xylitol using one or more of xylosereductase, L-arabitol dehydrogenase, ribitol dehydrogenase, ribitoltransporter, and L-xylulose reductase enzymes. The process does not haveto be performed by a microbial host. For example, enzymes could be addeddirectly to a substrate to convert the substrate to xylitol.

One embodiment of the invention provides a method for producingL-xylulose comprising fermenting a substrate comprising D-xylose; orL-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose andother sugars with one or more recombinant or isolated microorganisms ofthe invention, and collecting L-xylulose before it is converted toxylitol.

Xylitol Synthesis from L-Arabinose and D-Xylose: D-Xylose SpecificXylose Reductases

In one embodiment of the invention substrates comprising D-xylose; orL-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose andother sugars are converted to xylitol using a D-xylose-specific xylosereductase that can convert D-xylose to xylitol but cannot convertL-arabinose to L-arabitol. Such a pathway would allow the use ofinexpensive starting substrates (see e.g., Table 1). Furthermore, arecombinant microorganism host can be engineered to use the other sugarsin this material as carbon and energy sources thus increasing theoverall efficiency by simply deregulating the specific sugar degradationpathways. This disclosure represents the first report ofD-xylose-specific xylose reductases. All of the xylose reductasesdisclosed to date exhibit activity on both D-xylose and L-arabinose.

One embodiment of the invention provides a process for convertingsubstrates comprising D-xylose; or L-arabinose; or L-arabinose andD-xylose; or L-arabinose, D-xylose and other sugars to xylitol using aD-xylose-specific xylose reductase that can convert D-xylose to xylitolbut cannot convert L-arabinose to L-arabitol. The process does not haveto performed by a microbial host. For example, enzymes could be addeddirectly to a substrate to convert the substrate to xylitol.

In one embodiment of the invention a xylose-specific reductase is a P.stipitis XR gene that has the following mutations: Ser233Pro andPhe286Leu. This mutant can be improved by directed evolution usingiterative mutagenesis and screening for growth on media with increasingconcentrations of L-arabinose.

One embodiment of the invention provides an isolated microorganismcomprising xylose specific reductase activity, wherein the xylosespecific reductase activity does not convert L-arabinose to L-arabitol.A microorganism or process of the invention can produce an end-productof xylitol from a substrate comprising D-xylose; or L-arabinose; orL-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars. Nosubstantial amount of L-arabitol is produced as an end-product. Nosubstantial amount of L-aribitol is less than 10, 5, 2, or 1%L-arabitol. The substrate can be a xylan hydrolysate or hemicellulosehydrolysate. In one embodiment the substrate is an agricultural residuesuch as bagasse agricultural residue, corn cob agriculture residue, flaxstraw agricultural residue, wheat straw residue, oat hull agriculturalresidue, tree hydrolysate, or a combination thereof.

A microorganism can be a bacterium, such as E. coli, fungus or yeast. Inone embodiment, the microorganism is non-pathogenic.

A xylose-specific reductase can be encoded by a nucleic acid comprisinga P. stipitis xylose reductase (GenBank Acc. No. X59465) or a xylosereductase from another organism comprising a Ser233Pro mutation and aPhe286Leu mutation (SEQ ID NO:43) (FIG. 28).

The invention provides methods for producing xylitol comprisingfermenting a substrate comprising D-xylose; or L-arabinose; orL-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars withan isolated microorganism comprising xylose-specific reductase activity,wherein the xylose-specific reductase activity does not convertL-arabinose to L-arabitol. In one embodiment, the method does notrequire separation of L-arabitol from xylitol.

Xylitol Resistant Xylose Isomerases

To increase the tolerance of xylose isomerase to high levels of xylitol,an E. coli xylose isomerase (xylA) was mutagenisized. See, e.g., GenBankAccession Number K01996 and X04691. A microorganism with a xyloseisomerase with 1, 2, 3, 4, 5, 6, 7, 8 or more of the following mutationshas increased tolerance to xylitol: F9L, Q11K, S20L, L213Q, F283Y,K311R, H420N, H439Q. In particular, the following mutations can increasetolerance to xylitol:

-   -   (e) F9L, L213Q, F283Y, K311R, H420N;    -   (f) F9L, Q11K, L213Q, F283Y, K311R, H420N;    -   (g) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N; or    -   (h) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N, H439Q.

A microorganism with the mutated xylose isomerase can tolerate about 1%,2%, 5%, 8%, 10%, 15% or more xylitol. Preferably, the xylose isomeraseis purified. A purified protein is purified free of other components,such as other proteins, lipids, culture medium and polynucleotides. Forexample, the protein can be 50%, 75%, 90%, 95%, 96%, 97%, 98%, 99%, or100% purified. Proteins of the invention can comprise other peptidesequences, such as labels, linkers, signal sequences, TMR stop transfersequences, transmembrane domains, or ligands useful in proteinpurification such as glutathione-S-transferase, histidine tag, andstaphylococcal protein A.

One embodiment of the invention provides a nucleic acid moleculeencoding the mutant E. coli xylose isomerase described above. Anisolated nucleic acid is a molecule that is not immediately contiguouswith one or both of the 5′ and 3′ flanking genomic sequences that it isnaturally associated with. An isolated polynucleotide can be, forexample, a recombinant DNA or RNA molecule of any length, provided thatthe nucleic acid sequences naturally found immediately flanking therecombinant DNA or RNA molecule in a naturally-occurring genome isremoved or absent. Isolated nucleic acid molecules can benaturally-occurring or non-naturally occurring nucleic acid molecules. Anucleic acid molecule existing among hundreds to millions of othernucleic acid molecules within, for example, cDNA or genomic libraries,or gel slices containing a genomic DNA restriction digest are not to beconsidered an isolated polynucleotide.

Another embodiment of the invention provides a recombinant microorganismcomprising a mutated E. coli xylose isomerase coding sequence, whereinthe microorganism can grow in the presence of about 1% or more ofxylitol.

Other Approaches to the Production of Xylitol

Another approach to the production of xylitol is to convert anyL-arabitol formed into a readily metabolized substrate. This could beachieved by adding an L-arabinose to L-xylulose pathway to the hoststrain (FIG. 5) thus effectively removing any L-arabitol formed.

Another approach would be to use a biosynthetic pathway that does notuse a xylose reductase but an alternative enzyme system that does notform L-arabitol from L-arabinose. Such a route is outlined in FIG. 6, itutilizes a xylose isomerase coupled with xylitol dehydrogenase, and thisroute will form xylitol via a D-xylulose intermediate.

Screening Strains and Methods

The invention also provides screening methods that can be combined withdirected evolution to select D-xylose-specific reductases. The screeningmethod is outlined in FIG. 4 and takes advantage of the observation thatphosphorylated sugars are toxic to, for example, E. coli if allowed toaccumulate. See, Scangos & Reiner (1979). “A unique pattern of toxicsynthesis in pentitol catabolism: implications for evolution.” J MolEvol 12(3):189-95; Scangos & Reiner (1978). “Acquisition of ability toutilize Xylitol: disadvantages of a constitutive catabolic pathway inEscherichia coli.” J Bacteriol 134(2):501-5. To take advantage of such ascreen a kinase is required that can phosphorylate L-arabitol but notxylitol; the L-ribulokinase of E. coli is such an enzyme. See, Lee etal. (2001). “Substrate specificity and kinetic mechanism of Escherichiacoli ribulokinase.” Arch Biochem Biophys, 396(2):219-24.

The host strain can be auxotrophic for D-xylose and L-arabinoseutilization and be able to grow on xylitol (carry an xdh gene). Anxdh/araB operon plasmid can be constructed in which the genes areconstitutively expressed in a strain that is an araBAD/xylA doublemutant. Such a strain, carrying a xylose reductase and grown on amixture of L-arabinose and D-xylose can grow by forming xylitol fromD-xylose. However, the XR will also form L-arabitol that in turn will beconverted to L-arabitol 5-phosphate which is lethal when accumulatedinside the cell. This is a powerful screen for mutant xylitol reductasesthat cannot synthesize L-arabitol while simultaneously synthesizingxylitol.

Xylose Reductase Screening Strain. Screening strains for detection andenhancement of the individual enzymes are an important part of theinvention. An outline for a xylose reductase screening strain is shownin FIG. 2. In one embodiment of the invention, an E. coli strain, suchas K12, has a xylose isomerase deletion (xylAΔ) making it unable to growon D-xylose. E. coli cannot synthesize or utilize xylitol as a carbonsource and addition of a deregulated xylitol dehydrogenase gene intothis host strain enables growth on xylitol because the XDH will convertxylitol to D-xylulose, which can then be utilized via intermediarymetabolism. When the screening strain is transformed with a plasmidcarrying a putative xylose reductase gene it can be used to screen forXR reductase activity. That is, active clones when grown on a D-xyloseminimal medium will only grow if the D-xylose is converted to xylitol.These screening strains are very useful for cloning novel aldosereductases, preliminary screening of mutagenesis libraries, and can alsobe adapted into a high throughput plate screen for evolved reductases.

L-Arabitol 4-Dehydrogenase Screening Strain. E. coli K12 cannotefficiently utilize L-arabitol or L-xylulose as sole carbon and energysources See, Badia et al. (1991). “L-lyxose metabolism employs theL-rhamnose pathway in mutant cells of Escherichia coli adapted to growon L-lyxose.” J Bacteriol 173(16):5144-50. It cannot utilize L-arabitolbecause it does not possess the required degradation pathway that isfound in other enteric organisms. See, Reiner (1975). “Genes for ribitoland D-arabitol catabolism in Escherichia coli: their loci in C strainsand absence in K-12 and B strains.” J Bacteriol, 123(2):530. Conversely,while E. coli K12 carries all the genes required for L-xyluloseutilization it does not use this substrate because the degradation genesare found in two separate cryptic operons. See, Ibanez et al. (2000).“Role of the yiaR and yiaS genes of Escherichia coli in metabolism ofendogenously formed L-xylulose.” J Bacteriol 182(16): 4625-7; Yew &Gerlt (2002). “Utilization of L-ascorbate by Escherichia coli K-12:assignments of functions to products of the yjf-sga and yia-sgboperons.” J Bacteriol 184(1):302-6. FIG. 3 shows the logic behind thisscreen. A screening strain requires the deregulation of three genes lyxK(L-xylulose kinase), ulaE (L-xylulose 5-phosphate epimerase) and ulaF(L-ribulose 5-phosphate 4-epimerase). In one embodiment of the inventionall the genes are cloned into a plasmid under control of a constitutivepromoter. In another embodiment of the invention the yiaJ gene(repressor protein of the yia-sgb operon of E. coli) is deleted. Oncederegulated, growth on L-xylulose is possible but not growth onL-arbinitol. This strain can then be used to screen for L-arbinitol4-dehydrogenase activity because it confers the ability of growth onL-arbinitol on the screening strain. Such a strain would also be usefulfor preliminary screening of L-arbinitol 4-dehydrogenase mutagenesislibraries so null mutations could be easily eliminated.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

The methods and compositions described herein as presentlyrepresentative of preferred embodiments are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art, which areencompassed within the spirit of the invention, are defined by the scopeof the claims.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof', and “consisting of may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of

Markush groups or other grouping of alternatives, those skilled in theart will recognize that the invention is also thereby described in termsof any individual member or subgroup of members of the Markush group orother group.

Co-pending patent application U.S. Ser. No. ______, filed May 19, 2005,entitled “Microbial production of xylitol via a hexose phosphate and apentose phosphate intermediate” is incorporated herein by reference inits entirety.

EXAMPLES Example 1 Cloning and Preliminary Analysis of the PichiaStipitis Xylose Reductase Gene Expressed in E. Coli

A Pichia stipitis XR was cloned using primers designed from thepublished sequence (GenBank Acc. #X59465) by reverse transcriptase-PCR(RT-PCR). P. stipitis was grown overnight on YM media containing 1%D-xylose (w/v). Total RNA was isolated using the NucleoSpin RNA II kit(Promega). The gene was amplified using specific primers (SEQ ID NO:1and SEQ ID NO:2, Table 2) and the Access RT-PCR system (BD Biosciences,USA) with an Eppendorf Mastercycler PCR machine, using standardamplification parameters. The RT-PCR reaction yielded a single band bygel electrophoresis. The gene was restricted with KpnI and BamHI usingstandard conditions then ligated using a rapid DNA ligation kit (Takarav.2, Takara Miros Bio, USA) into the cloning and expression plasmidpTTQ18, restricted with the same enzymes to yield pZUC5 (FIG. 7). DNAsequencing (AGCT, Northbrook, Ill.) showed complete identity to thepublished sequence.

The P. stipitis XR expressed in E. coli was analyzed using aspectrophotometric assay. The assay monitors the conversion of D-xyloseto xylitol by measuring the loss of a nicotinimide cofactor (NADH) atA₃₄₀. The host strain for this analysis was ZUC25/pZUC5, an E. coliW3110 xylAΔ strain (ZUC25) transformed with pZUC5. A single colony ofZUC25/pZUC5 was inoculated into 2 mL LB broth supplemented withampicillin (200 mg/L). A control culture ZUC25/pTTQ18 (pZUC25transformed with pTTQ18) was grown treated in the same way. The cultureswere incubated overnight at 30° C. with shaking (200 RPM). A 1 mLaliquot of each culture was then diluted into 100 mL fresh LB media withampicillin (200 mg/L) and incubation was continued (30° C., 200 RPM)until the A₆₆₀ was 0.1. Each culture was induced with 1 mM IPTG andculture was further incubated for an additional 3 hrs. Cells wereharvested by centrifugation (2000×g's, 20 mins), and the media wasdecanted. The cells were stored at −20° C. until needed for prior tocell lysis.

The cells were lysed with 1 mL BugBuster protein extraction reagent(Novagen, USA) at 37° C. The bacterial cell debris was removed withcentrifugation (12,000×g's, 10 mins). The cell lysate was then kept cold(4° C.) during the brief period before the activity assay was performed.For the assay, D-xylose (100 mM, Sigma, USA) and NADH (15 mg/mL,Calbiochem, USA) stock solutions were prepared in 100 mM PIPES buffer(Sigma). To perform the assay, 100 μL cell lysate was mixed with 1 mLD-xylose and 10 μL NADH stock solutions. Activity was measured byfollowing the decrease in absorbance at 340 nm due to the reduction ofNADH, readings were taken every minute for 10 mins. The lysatecontaining the induced P. stipitis XR showed a 9.6-fold increase in theNADH loss as compared to the negative control. This increase in rates isevidence for a functionally expressed P. stipitis XR enzyme in an E.coli host.

ZUC25/pZUC5 and the control strain ZUC25/pTTQ18 were also tested fortheir ability to convert D-xylose to xylitol by in vivo bioconversion.Each strain was inoculated from a single colony into 2 mL M9 minimalmedia supplemented with glycerol (0.2% v/v) and ampicillin (200 mg/L)and incubated overnight at 30° C. with shaking (200 RPM). Afterincubation, an aliquot of each was diluted 100-fold into 10 mL of freshM9 minimal media contain glycerol (0.2% v/v) and ampicillin (200 mg/L)and continued incubation (30° C., 200 RPM) until the A₆₆₀ reached 0.1.The cultures were induced with isopropyl-β-D-thiogalactopyranoside(IPTG, 1 mM and D-xylose (1% w/v) and ampicillin (200 mg/L) were added.The cultures were incubated at 30° C. with shaking (200 RPM) andaliquots were removed at various time points after the IPTG induction.The samples were then analyzed by HPLC analysis using an Aminex HPX-87Pcolumn (BioRad, USA). After 24 hrs of induction, the P. stipitis XRdisplayed 10% conversion of D-xylose to xylitol thus proving that theenzyme was functionally expressed. As expected the control strain didnot accumulate any xylitol. This confirmed the enzymatic data and provesthat the P. stipitis XR is functionally expressed in E. coli.

Example 2 Cloning of the yafB Aldose Reductase from E. Coli K12

The Escherichia coli aldose reductase (putative XR) gene was clonedusing the annotated sequence of yafB (GenBank, Acc. #AE000129). The genewas cloned directly from the genomic DNA of E. coli K12 strain ER1793.The genomic DNA was isolated using a modified procedure of the Qiagenminiprep (Qiagen Inc., USA) kit using a 2 mL culture of ER1793 grownovernight in Lauria-Bertani (Miller) media (LB). The procedure differsfrom the standard procedure in the addition of a 5 min vortexing step ofthe DNA sample after addition of buffer 2. The modified procedure gave alarge distribution of DNA fragment sizes as seen by agarose gelelectrophoresis. The gene was amplified by PCR using specific primers(SEQ ID NO:3 and SEQ ID NO:4, Table 2) in an Eppendorf Mastercyler PCRmachine from genomic DNA using a FailSafe PCR cloning kit (Epicentre,USA). This reaction yielded a single band when visualized by agarose gelelectrophoresis. The amplified DNA fragment was restricted with EcoRIand BamHI using standard conditions then ligated using a rapid ligationkit (Takara v.2, Takara Miros Bio, USA) into the cloning and expressionplasmid pTTQ18, restricted with the same enzymes to yield pZUC19 (FIG.8). DNA sequencing (AGCT, Northbrook, Ill.) of the isolated clone showedcomplete identity to the published sequence.

Example 3 Cloning and Preliminary Analysis of the Candida Tenuis XyloseReductase Gene

The Candida tenuis XR was cloned using primers designed from thepublished sequence (GenBank Acc. #AF074484) by RT-PCR. C. tenuis wasgrown overnight on YM media containing 1% D-xylose (w/v). The total RNAwas isolated using the NucleoSpin RNA II kit (Promega, USA). The genewas amplified using specific primers (SEQ ID NO:5 and SEQ ID NO:6, Table2) and the Access RT-PCR system (BD Biosciences, USA) with an EppendorfMastercycler PCR machine using standard amplification parameters. TheRT-PCR reaction yielded a single band by gel electrophoresis. Theamplified fragment was restricted with EcoRI and BamHI using standardconditions then ligated using a rapid ligation kit (Takara v.2, TakaraMiros Bio, USA) into the cloning and expression plasmid pTTQ18,restricted with the same enzymes to yield pZUC30 (FIG. 9). DNAsequencing (AGCT, Northbrook, Ill.) showed complete identity to thepublished sequence.

To test the enzymatic activity of the cloned C. tenuis XR in vivo, ZUC25(E. coli W3110 xylAΔ) was transformed with pZUC30 by electroporation.The resultant strain, ZUC25/pZUC30 and control strain ZUC25/pTTQ18(ZUC25 transformed pTTQ18) were tested for their ability to convertD-xylose to xylitol by in vivo bioconversion. Each strain was inoculatedfrom a single colony into 2 mL M9 minimal media supplemented withglycerol (0.2% v/v) and ampicillin (200 mg/L) and incubated overnight at30° C. with shaking (200 RPM). After incubation, an aliquot of each wasdiluted 100-fold into 10 mL of fresh M9 minimal media contain glycerol(0.2% v/v) and ampicillin (200 mg/L) and continued incubation (30° C.,200 RPM) until the A₆₆₀ reached 0.1. The cultures were induced withisopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM and D-xylose (1% w/v)and ampicillin (200 mg/L) were added. The cultures were incubated at 30°C. with shaking (200 RPM) and aliquots were removed at various timepoints after the IPTG induction. The samples were then analyzed by HPLCanalysis using an Aminex HPX-87P column (BioRad, USA). After 24hrs postinduction, the C. tenuis XR strain (ZUC25/pZUC30) displayed 15%conversion of D-xylose to xylitol thus proving that the enzyme wasfunctionally expressed in E. coli. The control strain ZUC25/pTTQ18 didnot accumulate any xylitol.

Example 4 Cloning and Preliminary Analysis of the Trichoderma ReeseiL-Arabitol 4-Dehydrogenase Gene (lad1)

The T. reesei L-arbinitol-4-dehydrogenase gene was cloned using primersdesigned from the published sequence (GenBank Acc. #AF355628) by RT-PCR.T. reesei was grown overnight on YM media containing 1% L-arabinose(w/v). Total RNA was isolated using the NucleoSpin RNA II kit (Promega,USA)). The gene was amplified using specific primers (SEQ ID NO:7 andSEQ ID NO:8, Table 2) and the Access RT-PCR system (BD Biosciences, USA)with an Eppendorf Mastercycler PCR machine, using standard amplificationparameters. The RT-PCR reaction yielded a single band by gelelectrophoresis. The gene was restricted with EcoRI and BamHI usingstandard conditions then ligated using a rapid DNA ligation kit (Takarav.2, Takara Miros Bio, USA) into the cloning and expression plasmidpTTQ18, restricted with the same enzymes to yield pZUC6 (FIG. 10). DNAsequencing (AGCT, Northbrook, Ill.) showed complete identity to thepublished sequence.

To test the activity of the cloned T. reesei LAD1 gene, plasmid pZUC6was inserted into strain ZUC29 (E. coli E. coli BW255113/xylAΔ,Δ[araD-araB]567, xylAΔ, lacZ4787 (Δ)(::rrnB-3), lacIp-4000[lacIQ, rph-1,Δ(rhaD-rhaB)568) (pZUC29/pZUC6)^(˜). The host strain, ZUC29 can notutilize L-arabitol and can therefore be used to screen for synthesis ofL-xylulose from L-arabitol. A control strain, ZUC29 transformed withpTTQ18 was also made for comparison. Each strain was inoculated from asingle colony into 2 mL M9 minimal media supplemented with glycerol(0.2% v/v) and ampicillin (200 mg/L) and incubated overnight at 30° C.with shaking (200 RPM). After incubation, an aliquot of each was diluted100-fold into 10 mL of fresh M9 minimal media contain glycerol (0.2%v/v) and ampicillin (200 mg/L) and continued incubation (30° C., 200RPM) until the A₆₆₀ reached 0.1. The cultures were induced withisopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM and L-arabinose (1% w/v)and ampicillin (200 mg/L) were added. The cultures were incubated at 30°C. with shaking (200 RPM) and aliquots were removed at various timepoints after the IPTG induction. The samples were monitored forL-xylulose formation from L-arabitol, the reverse of the in vivoreaction by HPLC analysis using a Aminex HPX-87P column (BioRad, USA).After 24 hrs post induction, the T. reesei LAD1 strain (ZUC29/pZUC6)displayed a 2.7% conversion of L-arabitol to L-xylulose thus showingthat the enzyme was functionally expressed in E. coli. As expected thecontrol strain ZUC29/pTTQ18 did not accumulate any L-xylulose.

Example 5 Cloning and Preliminary Analysis of the Trichoderma ReeseiL-Xylulose Reductase Gene (lxr1)

The T. reesei L-xylulose reductase gene was cloned using primersdesigned from the published sequence (GenBank Acc #AF375616) by RT-PCR.T. reesei was grown overnight on YM media containing 1% L-arabinose(w/v). The total RNA was isolated using the NucleoSpin RNA II kit(Promega, USA)). The gene was amplified using specific primers (SEQ IDNO:9 and SEQ ID NO:10, Table 2) and the Access RT-PCR system (BDBiosciences, USA) with an Eppendorf Mastercycler PCR machine, usingstandard amplification parameters. The RT-PCR reaction yielded a singleband by gel electrophoresis. The gene was restricted with EcoRI andBamHI using standard conditions then ligated using a rapid DNA ligationkit (Takara v.2, Takara Miros Bio, USA) into the cloning and expressionplasmid pTTQ18, restricted with the same enzymes to yield pZUC7 (FIG.11). DNA sequencing (AGCT, Northbrook, Ill.) showed complete identity tothe published sequence.

To test the activity of the cloned T. reesei LXR gene, plasmid pZUC7 wasinserted into strain ZUC29 by electroporation. A control strain wasconstructed by electroporating ZUC29 with pTTQ18 expression vector. Eachstrain was inoculated from a single colony into 2 mL M9 minimal mediasupplemented with glycerol (0.2% v/v) and ampicillin (200 mg/L) andincubated overnight at 30° C. with shaking (200 RPM). After incubation,an aliquot of each was diluted 100-fold into 10 mL of fresh M9 minimalmedia contain glycerol (0.2% v/v) and ampicillin (200 mg/L) andcontinued incubation (30° C., 200 RPM) until the A₆₆₀ reached 0.1. Thecultures were induced with isopropyl-β-D-thiogalactopyranoside (IPTG, 1mM and xylitol (0.1% w/v) and ampicillin (200 mg/L) were added. Thecultures were incubated at 30° C. with shaking (200 RPM) and aliquotswere removed at various time points after the IPTG induction. Thesamples were monitored for L-xylulose formation from xylitol (thereverse of the in vivo reaction) by HPLC analysis using a Aminex HPX-87Pcolumn (BioRad, USA). After 46 hrs post induction, the T. reesei LAD1strain (ZUC29/pZUC7) there was an 11% conversion of xylitol toL-xylulose thus showing that the enzyme was functionally expressed in E.coli. As expected the control strain ZUC29/pTTQ18 did not accumulate anyL-xylulose.

Example 6 Cloning of the Trichoderma Reesei Xylitol Dehydrogenase Gene

The T. reesei xylitol dehydrogenase gene was cloned using primersdesigned from the published sequence (GenBank Acc. #AF428150) by RT-PCR.T. reesei was grown overnight on YM media containing 1% D-xylose (w/v).Total RNA was isolated using the NucleoSpin RNA II kit (Promega, USA)).The gene was amplified using specific primers (SEQ ID NO:11 and SEQ IDNO:12) and the Access RT-PCR system (BD Biosciences, USA) with anEppendorf Mastercycler PCR machine, using standard amplificationparameters. The RT-PCR reaction yielded a single band by gelelectrophoresis. The gene was restricted with EcoRI and BamHI usingstandard conditions then ligated using a rapid DNA ligation kit (Takarav.2, Takara Miros Bio, USA) into the cloning and expression plasmidpTTQ18, restricted with the same enzymes to yield pZUC31 (FIG. 12). DNAsequencing (AGCT, Northbrook, Ill.) showed complete identity to thepublished sequence.

Example 7 Construction of an L-Arabitol 4-Dehydrogenase/L-XyluloseReductase Operon

The lad1/lxr1 operon was constructed in the following way: The lxr1 genewas replicated by PCR using a 5′ forward primer (SEQ ID NO:15) and a 3′reverse primer (SEQ ID NO:16, Table 2) using an Advantage 2 PCR kit (BDBiosciences, USA) in an Eppendorf Mastercycler PCR machine, usingstandard conditions. The 5′ forward primer has an XbaI restriction siteand a consensus ribosome binding site (RBS) upstream of the lxr1 ATGstart codon. The 3′ reverse primer has a HindIII restriction site. Thereplicated fragment was restricted with XbaI and HindIII, and ligatedinto pZUC6 cut with the same restriction enzymes. The resultant plasmidwas named pZUC18 (FIG. 13).

The throughput of this operon could be improved by mutagenesis andscreening of this operon in an E. coli strain expressing a xdh gene.When plated with L-arbinitol as a sole carbon source only clones able toconvert L-arbinitol to xylitol will grow. Further enhancements could bedetected using a high throughput crossfeeding strain in an analogous wayusing plates containing L-arabitol. For fine tuning the genes involvedin L-xylulose metabolism such as but not limited to lyxK, ulaE and ulaFcould be removed.

Example 8 Construction of a Xylose Reductase/L-Arabitol 4-DehydrogenaseOperons (XR/LAD1 and yafB/LAD1)

Two XR/LAD1operons were constructed using the E. coli yafB and the P.stipitis XYL1 xylose reductases in combination with the lad1 gene fromT. reesei. The lad1 gene was replicated by PCR using a 5′ forward primer(SEQ ID NO:17) and a 3′ reverse primer (SEQ ID NO:18, Table 2) using anAdvantage 2 PCR kit (BD Biosciences, USA) in an Eppendorf MastercyclerPCR machine, using standard conditions. The 5′ forward primer containedan BamHI restriction site and a consensus RBS upstream of the lad1 ATGstart codon. The 3′ reverse primer carried a XbaI restriction site. Thereplicated fragment was restricted with BamHI and XbaI, and ligated intopZUC5 and pZUC19 cut with the same restriction enzymes. The resultantplasmids named pZUC20 (P. stipitis XR/LAD1) and pZUC21 (E. coliyafB/LAD1) can be seen in FIGS. 14 and 15.

Example 9 Construction of the Xylose Reductase./L-Arabitol4-Dehydrogenase/L-Xylulose Reductase Operons

The construction of the P. stipitis XR/LAD1/LXR1 and operon was achievedby replacing the 200 bp HindIII-PstI fragment of pZUC20 with the 1004bpHindIII- PstI fragment of pZUC18, to create plasmid pZUC24 (FIG. 16).The yafB/LAD1/LXR1 was similarly constructed by replacing the 200 bpHindIII-PstI fragment of pZUC21 with the 1004 bp HindIII-PstI fragmentof pZUC18, to create plasmid pZUC25 (FIG. 17).

Example 10 Cloning of the Gluconobacter Oxydans D-Xylose DehydrogenaseGene and its Use in D-Xylose Reductase Screening Strains

The xylitol dehydrogenase gene was cloned directly from the genomic DNAof Gluconobacter oxydans strain NRRL B-72 using the published sequence(Sugiyama et al., 2003. Biosci Biotechnol Biochem 67:584) by PCR.Primers SEQ ID NO:19 and SEQ ID NO:20 (Table 2) were used to amplify thegene sequence. The amplified fragment was cleaved with restrictionenzymes EcoRI and BamHI followed by ligation into expression vectorpTrp338 cut with the same enzymes, to form plasmid pZUC15 (FIG. 18).

Deletion of the xlyA gene from E. coli K12 was carried out using thepublished RED deletion protocol. Datsenko & Wanner. 2000. Proc. Natl.Acad. Sci. USA 97, 6640-6645. The primers for the deletion were SEQ IDNO:21 and SEQ ID NO:22 (Table 2). This protocol only works well instrains that cannot metabolize L-arabinose so the deletion was initiallymade in BW25113 (supplied with the RED deletion kit) and thentransferred to E. coli AB707 and W3110 by P1 transduction by selectionfor the inserted chloramphenicol acetyltransferase gene (cat) (Shortcourse in Bacterial Genetics: A Laboratory Manual and Handbook forEscherichia coli and Related Bacteria, Jeffrey H. Miller, Cold SpringHarbor Laboratory; 1^(st) Ed., (Jan. 15, 1992). The antibiotic gene wasthen removed by FLP-mediated excision following the published protocol.The resultant Cm^(s) xylAΔ strains were named ZUC24 (AB707; xylAΔ) andZUC25 (W3110; xylAΔΔ, λ IN[rrnD-rrnE]1, rph-1). The BW25113/xylAΔ strainwas similarly cured of the cat gene and the resultant strain was namedZUC29.

ZUC24 and ZUC25 were transformed with plasmid pZUC15 by electroporationand the resultant strains were named ZUC26 and ZUC27 respectively. Thephenotypic characteristics of these strains are shown in Table 3.Strains ZUC26 and ZUC27 can be used as a selection hosts for XR activity(cloned on a compatible plasmid) because strains carrying active XR'swill be able to synthesize xylitol, which in turn will be converted toD-xylulose by the XDH and thus allow growth on D-xylose as sole carbonsource. The utility of strain ZUC26 as a XR screening strain is shown inTable 4. The results show that while pZUC19 conferred growth on D-xyloseat both 30° C. and 37° C., pZUC5 was only active at 30° C. These resultsconfirm the synthesis of xylitol from both of these reductases.

An alternative screening strain, ZUC49 was also constructed bytransformation of ZUC25 with the T. reesei xdh clone pZUC31 (FIG. 13).The phenotype and genotype of this strain are shown Table 3. ZUC49 wastransformed with pZUC30 (C. tenuis XR) and tested for growth onD-xylose, the results (Table 4) showed that growth occurred at 30° C.but not at 37° C.

Example 11 L-Arabitol 4-Dehydrogenase Screening Strain

The genes for the deregulated L-xylulose pathway were obtained from E.coli using PCR. It was constructed in two stages (FIG. 19), firstly theL-xylulose kinase (lyxK) was isolated using PCR using primers SEQ IDNO:23 and SEQ ID NO:24. The fragment was cleaved with EcoRI and BamHIand ligated into pTrp338 cut with the same enzymes to form pZUC4. Thetwo remaining genes ulaE and ulaF were replicated as a natural operonusing primer SEQ ID NO:25 and SEQ ID NO:26. The fragment was cleavedwith BglII and NcoI then ligated into pZUC4 cleaved with BamHI and Ncolto form pZUC8. When transformed with pZUC6 (LAD1 clone) this plasmidconferred growth on L-arabitol whereas the host carrying pTTQ18 doesnot.

Example 12 Cloning and Analysis of the Ambrosiozyma Monospora L-XyluloseReductase Gene (Alx1)

The A. monospora L-xylulose reductase gene was cloned using primersdesigned from the published sequence (GenBank Acc. #AJ583159) by RT-PCR.A. monospora was grown overnight on YM media containing 2% L-arabinose(w/v). Total RNA was isolated using the RNeasy kit (Qiagen, USA). Thegene was amplified using specific primers (SEQ ID NO:27 and SEQ IDNO:28) and the One-Step RT-PCR kit (Qiagen, USA) with a DNA EnginePeltier Thermal Cycler PCR machine (MJ Research, USA), using standardamplification parameters. The RT-PCR reaction yielded a single band bygel electrophoresis. The gene was restricted with EcoRI and BamHI usingstandard conditions then ligated using the Quick Ligation kit (NewEngland Biolabs, USA) into the cloning and expression plasmid pTTQ18,restricted with the same enzymes to yield pATX101 (FIG. 20). DNAsequencing using the BigDyeTerminator v3.1 Cycle Sequencing kit (AppliedBiosystems, USA) and the ABI PRISM 3100 Genetic Analyzer (AppliedBiosystems) showed complete identity to the published sequence.

To test the activity of the cloned A. monospora alx1 gene, plasmidpATX101 was inserted into strain ZUC99 (lyxKΔ, Δ(araD-araB)567,lacZ4787(Δ)(::rrnB-3), lacIp-4000(lacIQ),λ-, rph-1, Δ(rhaD-rhaB)568,hsdR514) by electroporation (ZUC99/pATX101). A control strain, ZUC99transformed with pTTQ18 was also made for comparison. Each strain wasinoculated from a single colony into 3 mL LB media supplemented withampicillin (200 mg/L) and incubated overnight at 30° C. with shaking(250 RPM). After incubation, an aliquot of each was diluted 100-foldinto 20 mL of fresh LB media contain xylitol (1% w/v) and ampicillin(200 mg/L) and incubated (30° C., 250 RPM) for 2 hrs. The cultures wereinduced with isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM). Thecultures were incubated at 30° C. with shaking (250 RPM) and aliquotswere removed at various time points after the IPTG induction. Thesamples were monitored for L-xylulose formation from xylitol, thereverse of the in vivo reaction by HPLC analysis using an Aminex HPX-87Pcolumn (BioRad, USA). After 24 hrs post induction, the Alx1 strain(ZUC99/pATX101) displayed a 12% conversion of xylitol to L-xylulose thusshowing that the enzyme was functionally expressed in E. coli. Asexpected the control strain ZUC99/pTTQ18 did not accumulate anyL-xylulose.

Example 13 Construction and Analysis of an L-Arabitol4-Dehydrogenase/L-Xylulose Reductase Operon (Lad1/A1x1)

The Lad1/Alx1 operon was constructed in the following way: The alx1 genecloned in plasmid pATX101 was replicated by PCR using a 5′ forwardprimer (SEQ ID NO:29).and a 3′ reverse primer (SEQ ID NO:30) using a TaqDNA polymerase (Qiagen, USA) in a DNA Engine Peltier Thermal Cycler PCRmachine (MJ Research, USA), using standard conditions. The 5′ forwardprimer has a BamHI restriction site and a consensus ribosome bindingsite (RBS) upstream of the Alx1 ATG start codon. The 3′ reverse primerhas an XbaI restriction site. The replicated fragment was restrictedwith BamHI and XbaI, and ligated into pZUC6 cut with the samerestriction enzymes. The resultant plasmid was named pATX106 (FIG. 21).

To test the activity of the Lad1/Alx1 operon, plasmid pATX106 wasinserted into strain ZUC99 by electroporation (ZUC99/pATX106). Thestrain was inoculated from a single colony into 3 mL LB mediasupplemented with ampicillin (200 mg/L) and incubated overnight at 30°C. with shaking (250 RPM). After incubation, an aliquot of the culturewas diluted 100-fold into 20 mL of fresh LB media contain L-arabitol (1%w/v) and ampicillin (200 mg/L) and incubated (30° C., 250 RPM) for 2hrs. To induce the operon, isopropyl-β-D-thiogalactopyranoside (IPTG, 1mM) was added. The culture which was not induced with IPTG was used as anegative control. The cultures were incubated at 30° C. with shaking(250 RPM) and aliquots were removed at various time points after theIPTG induction. The samples were monitored for xylitol formation fromL-arabitol by HPLC analysis using an Aminex HPX-87P column (BioRad,USA). After 25 hrs post induction, the Lad1/Alx1 operon strain(ZUC99/pATX106) displayed a 6% conversion of L-arabitol to xylitol thusshowing that the operon was functionally expressed in E. coli (Table 5).As expected the control culture did not accumulate any L-xylulose andxylitol (Table 5).

Example 14 Construction and Analysis of a Xylose Reductase/L-Arabitol4-Dehydrogenase/L-Xylulose Reductase Operon (XR/Lad1/Alx1)

An XR/Lad1/Alx1 operon was constructed using the P. stipitis xylosereductase gene, the T. reesei L-arabitol 4-dehydrogenase gene and the A.monospora L-xylulose reductase gene. The XR gene cloned in plasmidpZUC20 was replicated by PCR using a 5′ forward primer (SEQ ID NO:31)and a 3′ reverse primer (SEQ ID NO:32) using a Taq DNA polymerase(Qiagen, USA) in a DNA Engine Peltier Thermal Cycler PCR machine (MJResearch, USA), using standard conditions. The 5′ forward primer has anucleotide sequence annealing to an upstream region of the tac promoterin a pTTQ18 plasmid. Both of the 5′ forward and 3′ reverse primers havea BamHI restriction site. The replicated fragment was restricted withBamHI, and ligated into pATX106 cut with the same restriction enzyme anddephosphorylated with the Antarctic phosphatase (New England Biolabs,USA). The resultant plasmid was named pATX112 (FIG. 22).

To test the activity of the XR/Lad1/Alx1 operon, plasmid pATX112 wasinserted into strain ZUC99 by electroporation (ZUC99/pATX112). Thestrain was inoculated from a single colony into 3 mL LB mediasupplemented with ampicillin (200 mg/L) and incubated overnight at 30°C. with shaking (250 RPM). After incubation, an aliquot of the culturewas diluted 100-fold into 20 mL of fresh LB media contain L-arabinose(1% w/v) and ampicillin (200 mg/L) and incubated (30° C., 250 RPM) for 2hrs. The culture was induced with isopropyl-β-D-thiogalactopyranoside(IPTG, 1 mM). The culture was incubated at 30° C. with shaking (250 RPM)and aliquots were removed at various time points after the IPTGinduction. The samples were monitored for xylitol formation fromL-arabinose by HPLC analysis using an Aminex HPX-87P column (BioRad,USA). After 24 hrs post induction, the XR/Lad1/Alx1 operon strain(ZUC99/pATX112) displayed a 2% conversion of L-arabinose to xylitol,showing that the recombinant bacterium acquires the ability to producexylitol from L-arabinose (Table 6).

Example 15 Cloning and Analysis of the Klebsiella Pneumoniae RibitolDehydrogenase Gene (RbtD)

The K. pnuemoniae ribitol dehydrogenase gene was cloned using primersdesigned from the published sequence of the K. aerogenes ribitoldehydrogenase gene (GenBank Acc. #M25606) by PCR. A genomic DNA of K.pneumoniae was obtained from the ATCC culture collection. The gene wasamplified using specific primers (SEQ ID NO:33 and SEQ ID NO:34) and aTaq DNA polymerase (Qiagen, USA) in a DNA Engine Peltier Thermal CyclerPCR machine (MJ Research, USA), using standard amplification parameters.The PCR reaction yielded a single band by gel electrophoresis. The genewas restricted with EcoRI and BamHI using standard conditions thenligated using the Quick Ligation kit (New England Biolabs, USA) into thecloning and expression plasmid pTTQ18, restricted with the same enzymesto yield pATX114 (FIG. 23). DNA sequence of the K. pnuemoniae ribitoldehydrogenase gene was analyzed using the BigDyeTerminator v3.1 CycleSequencing kit (Applied Biosystems, USA) and the ABI PRISM 3100 GeneticAnalyzer (Applied Biosystems). The deduced amino acid sequences betweenthe ribitol dehydrogenase genes from K. aergogenes (GenBank Acc.#M25606) and K. pnuemoniae are identical, although DNA sequences differby 4 nucleotides.

The K. pneumoniae RbtD expressed in E. coli was analyzed using aspectrophotometric assay. The assay monitors the oxidation of L-arabitolto L-xylulose by measuring the change in absorbance at 340 nm, whichoccurs as a nicotinamide cofactor NAD is reduced to NADH. PlasmidpATX114 was inserted into strain ZUC99 by electroporation(ZUC99/pATX114). A single colony of the strain was inoculated into 3 mLLB broth supplemented with ampicillin (200 mg/L). A control cultureZUC99/pTTQ18 was grown treated in the same way. The cultures wereincubated overnight at 30° C. with shaking (250 RPM). An aliquot of eachculture was then diluted 100-fold into 3 mL fresh LB media withampicillin (200 mg/L) and incubation was continued (37° C., 250 RPM) for2hrs. Each culture was induced with 1 mM IPTG and culture was furtherincubated for an additional 6 hrs. Cells were harvested from 500 μLaliquot of each culture by centrifugation (14,000×g, 10 mins), and themedia was decanted. The cells were stored at −20° C. until needed forprior to cell lysis.

The cells were lysed with 50 μL BugBuster protein extraction reagent(Novagen, USA) at room temperature. The bacterial cell debris wasremoved with centrifugation (14,000×g, 20 mins). The cell lysate wasthen kept on ice during the brief period before the activity assay wasperformed. To perform the enzyme reaction, 10 μL cell lysate was mixedwith 990 μl reaction mixture (100 mM Tris-Cl (pH9.0), 0.5 mM MgCl₂, 2 mMNAD and 100 mM L-arabitol) in a quartz cuvette at 30° C. Activity wasmeasured by following the increase in absorbance at 340 nm, using aspectrophotometer (model 8453, Agilent, USA). Protein amount in thelysate was determined using the DC Protein Assay kit (BioRad, USA),using bovine serum albumin for standard curve construction. One unit wasdefined as the amount of enzyme that caused the reduction of 1.0 μmolNAD to NADH per min. The lysate containing the induced K. pneumoniaeRbtD showed 0.65 unit/mg protein. The lysate from the control strain didnot show any activity.

ZUC99/pATX114 was also tested for their ability to convert L-arabitol toL-xylulose by in vivo bioconversion. The strain was inoculated from asingle colony into 3 mL LB media supplemented with ampicillin (200 mg/L)and incubated overnight at 30° C. with shaking (250 RPM). Afterincubation, an aliquot of the culture was diluted 100-fold into 3 mL offresh LB media contain L-arabitol (1% w/v) and ampicillin (200 mg/L) andincubated (37° C., 250 RPM) for 2 hrs. The culture was induced withisopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM). The culture wasincubated at 37° C. with shaking (250 RPM) and aliquots were removed atvarious time points after the IPTG induction. The samples were monitoredfor L-xylulose formation from L-arabitol by HPLC analysis using anAminex HPX-87P column (BioRad, USA). After 24 hrs post induction, theRbtD strain (ZUC99/pATX114) displayed a 19% conversion of L-arabitol toL-xylulose (Table 7).

These results clearly show that the ribitol dehydrogenase RbtD isfunctionally expressed in E. coli and can convert L-arabitol toL-xylulose.

Example 16 Construction and Analysis of a Ribitol Dehydrogenase/RibitolTransporter Operon (RbtD/RbtT)

The RbtD/RbtT operon was constructed in the following way: The K.pneumoniae ribitol transporter RbtT gene was isolated by PCR usingprimers designed from the published sequence (GenBank Acc. #AF045244). Agenomic DNA of K. pneumoniae was obtained from the ATCC culturecollection. The gene was amplified using specific primers (SEQ ID NO:35and SEQ ID NO:36) and the PfuUltra Hotstart DNA polymerase (Stratagene,USA) in a DNA Engine Peltier Thermal Cycler PCR machine (MJ Research,USA), using standard amplification parameters. The 5′ forward primer hasa BamHI restriction site and a consensus ribosome binding site (RBS)upstream of the RbtT ATG start codon. The 3′ reverse primer has an XbaIrestriction site. The replicated fragment was restricted with BamHI andXbaI, and ligated into pATX114 cut with the same restriction enzymes.The resultant plasmid was named pATX115 (FIG. 24).

To test the activity of the RbtD/RbtT operon, plasmid pATX115 wasinserted into strain ZUC99 by electroporation (ZUC99/pATX115). Thestrain was inoculated from a single colony into 3 mL LB mediasupplemented with ampicillin (200 mg/L) and incubated overnight at 30°C. with shaking (250 RPM). After incubation, an aliquot of the culturewas diluted 100-fold into 3 mL of fresh LB media contain L-arabitol (1%w/v) and ampicillin (200 mg/L) and incubated (37° C., 250 RPM) for 2hrs. The culture was induced with isopropyl-β-D-thiogalactopyranoside(IPTG, 1 mM). The culture was incubated at 37° C. with shaking (250 RPM)and aliquots were removed at various time points after the IPTGinduction. The samples were monitored for L-xylulose formation fromL-arabitol by HPLC analysis using an Aminex HPX-87P column (BioRad,USA). After 24 hrs post induction, the RbtD/RbtT operon strain(ZUC99/pATX115) displayed a 57% conversion of L-arabitol to L-xyluloseshowing that the ribitol transporter RbtT improved the in vivobioconversion of L-arabitol to L-xylulose (Table 7).

Example 17 Construction and Analysis of a Ribitol Dehydrogenase/RibitolTransporter/L-Xylulose Reductase Operon (RbtD/RbtT/Alx1)

The RbtD/RbtT/Alx1 operon was constructed in the following way: The Alx1gene in pATX101 was replicated by PCR using a 5′ forward primer (SEQ IDNO:37) and a 3′ reverse primer (SEQ ID NO:30) using the PfuUltraHotstart DNA polymerase (Stratagene, USA) in a DNA Engine PeltierThermal Cycler PCR machine (MJ Research, USA), using standardconditions. The 5′ forward primer has a nucleotide sequence annealing toan upstream region of the tac promoter in a pTTQ18 plasmid. Both of the5′ forward and 3′ reverse primers have an XbaI restriction site. Thereplicated fragment was restricted with XbaI, and ligated into pATX115cut with the same restriction enzyme and dephosphorylated with theAntarctic phosphatase (New England Biolabs, USA). The resultant plasmidwas named pATX118 (FIG. 25).

To test the activity of the RbtD/RbtT/Alx1 operon, plasmid pATX118 wasinserted into strain ZUC99 by electroporation (ZUC99/pATX118). Thestrain was inoculated from a single colony into 3 mL LB mediasupplemented with ampicillin (200 mg/L) and incubated overnight at 30°C. with shaking (250 RPM). After incubation, an aliquot of the culturewas diluted 100-fold into 20 mL of fresh LB media contain L-arabitol (1%w/v) and ampicillin (200 mg/L) and incubated (30° C., 250 RPM) for 2hrs. The culture was induced with isopropyl-β-D-thiogalactopyranoside(IPTG, 1 mM). The culture was incubated at 30° C. with shaking (250 RPM)and aliquots were removed at various time points after the IPTGinduction. The samples were monitored for xylitol formation fromL-arabitol by HPLC analysis using an Aminex HPX-87P column (BioRad,USA). The RbtD/RbtT/Alx1 operon strain (ZUC99/pATX118) exhibited 23% and39% conversions of L-arabitol to xylitol after 25 hrs and 49 hrs postinduction, respectively (Table 8).

Example 18 Construction of a Screening Strain for Selecting D-XyloseReductases that are Specific for D-Xylose Reduction

The xylA, araBADΔ host strain required for the screen (ZUC29) wasconstructed as described in example 10. A xdh/araB operon wasconstructed by as shown in FIG. 26. The araB gene was amplified by PCRusing primers SEQ ID NO:38 and SEQ ID NO:39 (Table 2) using E. coli K12chromosomal DNA as a template. The fragment was digested with BglII/NcoIand ligated into plasmid pZUC15 cleaved with BamHI/NcoI to yield pZUC22.ZUC29 was transformed with pZUC22 to complete the screening strain andwas named ZUC41. This strain when transformed with pZUCS (P. stipitis XRclone) cannot grow on D-xylose/L-arabinose mixtures (Table 9) due toL-arabitol 5-phosphate toxicity and as such can be used to screen forD-xylose specific xylose reductases. This could be achieved bysubjecting a cloned XR gene to one or multiple rounds of mutagenesisfollowed by selection on plates containing D-xylose and L-arabinose.Only mutants that can convert D-xylose to xylitol but not L-arabinose toL-arabitol will be able to grow.

Example 19 Selection of C. Tenuis XR Mutants Functional at 37° C. UsingXR Screening Strain pZUC49

The C. tenuis XR gene was mutated using the GeneMorph II error-prone PCRkit following the manufacturers protocol (Stratagene, USA). Genelibraries were screened in strain ZUC49 (see Example 10), selection wasfor growth on M9 minimal medium plates containing D-xylose as solecarbon source at 37° C. A mutant, which could now grow at 37° C. wasisolated and shown to confer growth at 37° C. when reintroduced to ZUC49by electroporation, whereas the w.t. clone pZUC30 did not. Sequencing ofthe mutated gene showed two changes from the w.t. gene, glycine 32 waschanged to a serine (Gly32Ser) and asparagine 138 was changed to anaspartate (Asn138Asp) (SEQ ID NO:44) (FIG. 29).

Example 20 Selection of a D-Xylose Specific XR Reductase

The P. stipitis XR gene was mutated using the GeneMorph II error-proneper kit following the manufacturers protocol (Stratagene, USA). Genelibraries were screened in strain ZUC41 (see Example 12), selection wasfor growth on M9 minimal medium containing 0.2% D-xylose and the minimalamount of L-arabinose that was found to be inhibitory, 0.001%. A plasmidlinked mutant was obtained that conferred enhanced growth in thepresence of 0.001% was obtained. Sequencing of the mutated XR geneshowed two mutations, Ser233Pro and Phe286Leu. This mutant has thepotential to be improved by directed evolution using iterativemutagenesis and screening for growth on media with increasingconcentrations of L-arabinose.

Example 21 Construction of a Xylitol Dehydrogenase Xylose Isomerase(xdh/xylA) Operon and its use in Synthesizing Xylitol from D-Xylose Viaa D-Xylulose Intermediate

An xdh/xylA operon was constructed as shown in FIG. 27. The xylAfragment was generated by PCR using primers SEQ ID NO:13 and SEQ IDNO:14, using E. coli K12 chromosomal DNA as a template. The fragment wasdigested with BamHI/NcoI and ligated into pZUC31 restricted with thesame enzymes and two independent clones were isolated. The resultantplasmids were named pZUC35 and pZUC36.

To test for synthesis of xylitol using D-xylose as starting substrate(see FIG. 6 for rationale) a host that cannot utilize D-xylose orD-xylulose is favored, e.g. a xylAB mutant. ZUC22 (E. coli K12prototroph AB707, xylAΔ::cam) is such a mutant. In this strain the xylAgene has been replaced with a chloramphenicol (cam) resistance gene, thecam gene has a polar effect on the xylB (D-xylulose kinase) genedownstream of xylA. As such this strain cannot utilize either D-xyloseor D-xylulose. ZUC22 was transformed with pZUC35 and pZUC36 byelectroporation to form strain ZUC53 and ZUC54. A control strainconsisting of ZUC22 transformed with expression vector pTrp338 was alsoconstructed and named ZUC52.

The conversion of D-xylose to xylitol was tested in the following way;100 ml of

M9 minimal medium was made up containing 0.25% glycerol and 0.361%D-xylose, 25 ml aliquots were dispensed into three sterile 100 ml baffleflasks. The flasks were inoculated with 0.25 ml an overnight culture ofZUC52, 53 and 54 grown in LB medium plus 40 ug/ml kanamycin. The flaskswere incubated for 24 hr at 37° C. with shaking (250 rpm). Samples weretaken after 24 hr then analyzed by HPLC analysis using an Aminex HPX-87Pcolumn (BioRad, USA). The results show, that both ZUC53 and ZUC54exhibited a 65% and 68% conversion of D-xylose to xylitol, whereas thecontrol strain had only a 0.002% conversion (Table 10). This result isunexpected because xylitol dehydrogenase is a catabolic enzyme and isfavored in the conversion of xylitol to D-xylulose. One would thereforeexpect the reaction to reach equilibrium between xylitol, D-xylose andD-xylulose, which it clearly does not.

Example 22 Selection of Sugar Transport Host ZUC72 for Xylitol Synthesisfrom Hemicellulose

It has previously been shown that E. coli strains carrying a ptsGmutation are relieved of catabolite repression and that such strains cansimultaneously grow on a wide range of sugars in the presence ofglucose. See, Kimata et al. (1997). “cAMP receptor protein-cAMP plays acrucial role in glucose-lactose diauxie by activating the major glucosetransporter gene in Escherichia coli.” Proc Natl Acad Sci, 94(24):12914-12919; Nichols & Dien et al. (2001). “Use of catabolite repressionmutants for fermentation of sugar mixtures to ethanol.” Appl MicrobiolBiotechnol, 56(1-2):120-5.

Such a strain would have a two fold benefit for xylitol synthesis fromhemicellulose hydrolysate as one can easily construct a strain that doesnot grow on D-xylose (the precuror to xylitol) but transports itefficiently while being able to co-utilize glucose and other sugarsfound in the hemicellulose hydrolyzate. Such a strain was constructed asfollows:

-   -   1. A phage P1 ptsG tranducing lysate was obtained by growing P1        on E. coli strain ND15(Nichols, Dien et al. 2001) using standard        techniques (Short course in Bacterial Genetics: A Laboratory        Manual and Handbook for Escherichia coli and Related Bacteria,        Jeffrey H. Miller, Cold Spring Harbor Laboratory; 1^(st) Ed.,        (Jan. 15, 1992). The ptsG mutation is closely linked to the        tetracycline resistance (tet^(R)) gene of transposon Tn10.    -   2. E. coli AB707 (prototroph) was transduced to tet^(R) and ptsG        mutants were identified as blue colonies when plated on medium        containing glucose, lactose and the chromogenic substrate X-gal,        the strain was named ZUC56.    -   3. ZUC56 was transduced to Kan^(R) using phage P1 grown on        BW25113 xylB::kan to yield ZUC58.    -   4. ZUC58 was passaged several times in M9 glucose minimal medium        plus kanamycin (50 mg/L) to select for enhanced glucose        utilizers. The fastest growing variant was isolated, purified        and named ZUC70.        The kanamycin gene was excised from ZUC70 using FLP mediated        excision to yield ZUC72. Datsenko & Wanner (2000). “One-step        inactivation of chromosomal genes in Escherichia coli K-12 using        PCR products.” Proceedings of the National Academy of Sciences        of the United States of America, 97(12): 6640-6645. ZUC72 can        grow efficiently on L-arabinose and glucose simultaneously but        cannot utilize D-xylose (Table 11).

Example 23 Mutagenesis and Selection of Xylitol Resistant XyloseIsomerases

The GeneMorph II kit (Stratagene) was used to generate error-prone PCRlibrary following the standard procedure to generate approximately 1-10mutations per 1000 base pairs. The E. coli xylose isomerase gene (xylA)was initially cloned into the pTRP338-H3 expression plasmid usingBamHI/NcoI restriction sites. DNA primers SEQ ID NO: 40 and SEQ ID NO:41 (Table 2), were used to amplify the xylA gene using a GeneMorph IIkit. The randomly mutated gene was the digested with restriction enzymesand ligated into freshly prepared pTRP338-H3 plasmid. This library wasinitially transformed into EC100 cells (Epicentre), plated onto richgrowth media containing kanamycin (50 mg/L), and incubated overnight at37° C. The resulting transformants were then scraped form from theplates and resuspended in fresh L-broth. The cells were pelleted bycentrifugation, and the plasmid library was extracted using a HiSpeedmidiprep plasmid isolation kit (Qiagen). This extra step in preparingthis plasmid library was necessary to create a high concentration ofplasmid DNA that was more suitable for transformation into the E. coliselection strain (ZUC29). Selection strain ZUC29 was then transformedwith the mutated plasmid library using standard electroporationprocedures. After phenotypic expression of the plasmid encoded kanamycinresistance gene, the cells were washed with 1×M9 salts to remove anyresidual rich growth media. The cells were then resuspended in 1×M9salts and plated onto minimal M9 media containing kanamycin (50 mg/L),0.2% D-xylose (w/v), and up to 10% xylitol (w/v). The plates wereincubated at 37° C. until colonies appeared, usually 2-3 days.

The fast growing colonies were picked and restreaked onto freshselection media plates and incubated at 37° C. The restreaking step isnecessary to remove contaminating slower growing colonies from theoriginal selection plate. Plasmid DNA was extracted from fast growingisolates from the second selection. ZUC29 was then transformed with theputative xylitol resistant candidates and screened again for growth onD-xylose/xylitol selection plates. Plasmids that transferred the xylitolresistant phenotype (XTL^(R)) were DNA sequenced (ACGT, Inc.) andcompared to the w.t. DNA sequence, the differences in the translation ofthe mutants versus the w.t. sequence was then determined (Table 12).This cycle was repeated twice using the best isolate from each round ofmutagenesis as the parent for the next round to produce mutant #3 thatcould grow in the presence of 3% xylitol.

Mutant #3 was further mutagenized using the XL1-RED mutagenesis kit(Stratagene). Mutant #8 was further mutagenized by error prone per(GeneMorph II, Stratagene) and a mutant was selected that was able togrow in the presence of 10% xylitol (Table 12), the gene had oneadditional mutation H439Q. Finally, the mutated xylA was cloned behindthe xdh gene of pZUC31 using a BamHI/NcoI digest to form pZUC52 prior tofermentation testing (similar construction as shown in FIG. 21).

Example 24 Fermentation of W.T. xylA and Mutant xylA10% using 5%D-Xylose as Substrate

ZUC72 was transformed with pZUC36 (FIG. 21) and pZUC52 to yield strainsZUC73 and ZUC112 respectively. The strains were tested for theconversion of D-xylose to xylitol in 1 L BIOSTAT®B fermenters (B. Braun)under the following conditions:

Growth Medium g/L Tryptone 10 Yeast extract 5 Potassium phosphate,dibasic 3 Potassium phosphate monobasic 2 Sodium chloride 5 Magnesiumsulfate 1 Water to 925 ml

The vessels were sterilized with the above media in situ, D-xylose (70 gin 175 ml) and D-glucose (60 g in 150 ml) was sterilized separately.Preinoculation, 100 ml of D-xylose and 20 ml of D-glucose feed was addedto the vessel. The fermenters were inoculated with 50 ml of an overnightstarter culture grown in LB at 37° C. and run under the followingconditions:

Temperature 37° C. pH 7.0 (NAOH control) Air 2 LPM (2 VVM) FeedD-xylose: 75 ml, 6-22 hr D-glucose: 130 ml, 8-46 hr Agitation 1200Samples were taken at regular time intervals and analyzed by HPLC. Theresults show that the xylitol resistant xylose isomerase (XI^(10%))produced 3.3% xylitol after 30 hr as compared to the w.t. XI whichproduced only 1.8% in the same amount of time. This represents a 54%increase in xylitol titer of the fermentation.

Example 25 Synthesis of Xylitol by ZUC112 Using 10% Under High D-XyloseConditions

Fermentations were run as follows:

Bacto Tryptone  10 g Bacto Yeast extract   5 g Potassium phosphate,dibasic   3 g Potassium phosphate monobasic 1.5 g Sodium chloride   5 gMagnesium sulfate.7H2O   1 g Cognis BioSpumex 36K antifoam~4 drops Waterto 750 mL

The vessels were sterilized with the above media in situ, D-xylose (100g) and D-glucose (10 g) was sterilized in 170 ml water separately andadded prior to inoculation of the vessel. A D-xylose (100 g), D-glucosefeed (60 g) was dissolved in 270 ml water, sterilized and used to feedthe fermentation to keep the D-xylose concentration ˜8%. The fermenterswere inoculated with 50 ml of an overnight starter culture grown in LBat 37° C. and run under the following conditions:

Temperature 37° C. pH 7.0 (NaOH control) Air 2 LPM (2 VVM) FeedD-xylose/D-glucose: 277 ml, 13-33 hr Agitation 1200 Volume afterinoculation  970 ml Final Volume (70 hr) 1105 mlUnder high xylose conditions a maximum of 7.2% xylitol (72 g/L) wassynthesized from 200 g of D-xylose or 79.5 g total when allowing fordilution due to feeding (final volume 1105 ml).

TABLE 1 Examples of Various Sugars in Agricultural Residues (% dryweight) Residue D-Xylose (%) L-Arabinose (%) D-Glucose (%) Bagasse 60 1525 Corn Cobs 65 10 25 Flax Straw 65 13 1 Wheatstraw 58 9 28

TABLE 2 List of DNA PCR primers. Enzyme Organism Forward PrimerReverse Primer XR P. stipitis SEQ ID NO: 1 SEQ ID NO: 2GTGTGTGTCATATGCCTTCTA GTGTGGATCCTTAGACGAAG TTAAGTTGAACT ATAGGAATCTTGTCXR E. coli K12 SEQ ID NO: 3 SEQ ID NO: 4 GTGTGAATTCGATGGCTATCCCACAGGATCCCTAATCCCATT CTGCATTTGG CAGGAGCCA XR C. tenuis SEQ ID NO: 5SEQ ID NO: 6 GAGAGAATTCGATGAGCGCA GAGAGGATCCTTAAACGAAG AGTATCCCAGACATTCGAATGTTGTC LAD1 T. reesei SEQ ID NO: 7 SEQ ID NO: 8GTGTGAATTCGATGTCGCCTT GTGTGGATCCTCAATCCAGGC CCGCAGTCGA TCTGAATCATGAC LXRT. reesei SEQ ID NO: 9 SEQ ID NO: 10 GTGTGAATTCGATGCCTCAGCGTGTGGATCCTTATCGTGTAG CTGTCCCCAC TGTAACCTCCGTC XDH T. reeseiSEQ ID NO: 11 SEQ ID NO: 12 GTGTGAATTCGATGGCGACTC CACAGGATCCTTACACCTTCTAAACGATCAAC CGTTGGGCC XylA E. coli K12 SEQ ID NO: 13 SEQ ID NO: 14TATAAGCTTAAGGAGGATCC TCGAAGCTTAGATCTCCATGG ATTATGGAGTTCAA TTATTTGTCGAACLXR1 T. reesei SEQ ID NO: 15 SEQ ID NO: 16 TGCTCTAGATAAGGAGGATATGCTCTAGATAAGGAGGATA ATAAATGCCTCAGCCTGTCCC ATAAATGCCTCAGCCTGTCCC CAC CACLAD1 T. reesei SEQ ID NO: 17 SEQ ID NO: 18 TCGGATCCTAAGGAGGATATAGCTCTAGATCAATCCAGGCT ATAATGTCGCCTTCCGCAGTC CTGAATCATGAC GATG XDHG. oxydans SEQ ID NO: 19 SEQ ID NO: 20 CAGCGATGAATTCGAAGAAGAGCGGATCCTTAACCGCCAGC AATCGGC XylA E. coli K12 SEQ ID NO: 21SEQ ID NO: 22 RED CCAATATTACGACATCATCCA TACCGATAACCGGGCCAACG deletionTCACCCGCGGCATTACCTGGT GACTGCACAGTTAGCCGTTAC GTAGGCTGGAGCTGCTTCATATGAATATCCTCCTTAG LyxK E. coli SEQ ID NO: 23 SEQ ID NO: 24AGCGAATTCATGACGC ATCGGATCCTTATAATGTGTG AATACTGGCTGG CTCCTTAATGC UlaE/FE. coli SEQ ID NO: 25 SEQ ID NO:26 TCTAGATCTAATATGTTGTCCGCACCATGGTTACTTCTGCCC AAACAAATCC GTAATAAG ALX1 A. SEQ ID NO: 27SEQ ID NO: 28 monospora GCGAATTCGATGACTGACT GAGGGATCCCTACCAAGAACATTCCAAC AGTGAAACC ALX1 A. SEQ ID NO: 29 SEQ ID NO: 30 monosporaGCGGATCCATAAAGGAGG GCTCTAGACTACCAAGAA ATATATAATGACTGACTACGTGAAACCACCATCAAC ATTCC XR P. stipitis SEQ ID NO: 31 SEQ ID NO: 32GCGGATCCCGACATCATAA GCGGATCCTTAGACGAAG CGGTTC ATAGGAATCTTGTC RBTD K.SEQ ID NO: 33 SEQ ID NO: 34 pneumoniae GCGGAATTCGATGAAGCACGGGATCCTCAGAGATCCA CTCTGTCTCCTC CGCTGTTC RBTT K. SEQ ID NO: 35SEQ ID NO: 36 pneumoniae GCGGATCCTAAGGAGGAT GCTCTAGATTAAGACTCTGATATTATGTCCGTTAATAA CCGCGTTG CAAA C ALX1 A. SEQ ID NO: 37 SEQ ID NO: 30monospora GCCTCTAGACGACATCATA GCTCTAGACTACCAAGAA ACGGTTCTGGTGAAACCACCATCAAC AraB E. coli SEQ ID NO: 38 SEQ ID NO: 39TTCAGATCTAACGATGGCGAT GCACCATGGTTATAGAGTCGC TGC AACGGCCTG pTRP200-SEQ ID NO: 40 seq-forw CGAACTAGTTAACTTTTACGC primer AAGT pTRP338-SEQ ID: 41 seq-rev GGCTGAAAATCTTCTCTCATC primer C

TABLE 3 Growth Phenotypes of XR Screening Strains. Growth at 30° C. and37° C. Strain Plasmid/Gene Genotype D-Glu D-Xylose Xylitol ZUC24 xylAΔYes No No ZUC25 xylAΔ, λ-, Yes No No IN[rrnD-rrnE]1, rph-1 ZUC26pZUC15Ixdh xylAΔ Yes No Yes ZUC27 pZUC15Ixdh xylAΔ, λ-, Yes No YesIN[rrnD-rrnE]1, rph-1 ZUC49 pZUC31/xdh xylAΔ, λ-, Yes No YesIN[rrnD-rrnE]1, rph-1

TABLE 4 Utility of XR Screening Strains. Relevant Growth at 30° C.Strain Host Strain Plasmid/Gene Genotype D-Glu D-Xylose ZUC26 ZUC24pZUC15/xdh xylAΔ Yes No ZUC31 ZUC24 pZUC15/xdh + xylAΔ Yes No pTTQ18control ZUC32 ZUC24 pZUC15/xdh + xylAΔ Yes Yes pZU19/yafB ZUC27 ZUC24pZUC15/xdh + xylAΔ Yes Yes¹ pZUC5/XR ZUC49 ZUC25 PZUC31/XR xylAΔ Yes NoZUC50 ZUC49 pZUC31/xdh + xylAΔ Yes Yes¹ pZUC30/XR ¹No growth at 37° C.,i.e. both xylose reductases temperature sensitive.

TABLE 5 Conversion of L-Arabitol to Xylitol via an L-XyluloseIntermediate. L-Arabitol (g/L) L-Xylulose (g/L) Xylitol (g/L) % StrainIPTG 0 hr 25 hrs 0 hr 25 hrs 0 hr 25 hrs Con¹⁾ ZUC99/pATX106 + 10.4 8.73± 0.13 0 0.57 ± 0.05 0 0.64 ± 0.03 6.2 ZUC99/pATX106 − 10.4 10.1 ± 0.010 0 0 0 0 ¹⁾ % conversion of L-arabitol to xylitol in 25 hrs.

TABLE 6 Conversion of L-Arabinose to Xylitol via an L-Arabitol andL-Xylulose Intermediates. L-Arabinose (g/L) L-Arabitol (g/L) L-Xylulose(g/L) Xylitol (g/L) % Strain 0 hr 24 hrs 0 hr 24 hrs 0 hr 24 hrs 0 hr 24hrs Con¹⁾ ZUC99/pATX112 9.96 5.57 ± 0.23 0 4.69 ± 0.32 0 0 0 0.17 ± 0.081.7 ¹⁾ % conversion of L-arabinose to xylitol in 24hrs.

TABLE 7 Conversion of L-Arabitol to L-Xylulose using RibitolDehydrogenase with or without Ribitol Transporter. L-Arabitol (g/L)L-Xylulose (g/L) % Strain 0 hr 24 hrs 0 hr 24 hrs Conversion¹⁾ ZUC99/10.94 7.75 ± 0.09 0 2.09 ± 0.16 19 pATX114 ZUC99/ 10.94 2.78 ± 0.19 06.28 ± 0.50 57 pATX115 ¹⁾% conversion of L-arabitol to L-xylulose in 24hrs.

TABLE 8 Conversion of L-Arabitol to Xylitol using RbtD/RbtT/Alx1 Operon.L-Arabitol (g/L) L-Xylulose (g/L) Xylitol (g/L) % Conversion¹⁾ Strain 0hr 25 hrs 49 hrs 0 hr 25 hrs 49 hrs 0 hr 25 hrs 49 hrs 25 hrs 49 hrsZUC99/ 11.9 8.26 ± 0.64 3.01 ± 0.37 0 1.00 ± 0.07 3.46 ± 0.27 0 2.76 ±0.14 4.58 ± 0.16 23 39 pATX118 ¹⁾ % conversion of L-arabitol to xylitolin 25 hrs or 49 hrs.

TABLE 9 Toxicity of L-Arabinose in the Presence of Xylose ReductaseActivity. Growth at 30° C. 0.2% D- Strain/ Relevant Plasmid 0.2% 0.2% D-xylose + Plasmid Genotype Genes Glucose¹ xylose¹ 0.2% L-ara¹ ZUC29/Δ(araD-araB) xdh, Yes Yes No² pZUC22 + 567, xylAΔ araB, PZUC5 xr ZUC29/A(araD-araB) xr Yes No² No² pZUC5 567, xylAΔ ¹M9 minimal medium + 1 mMIPTG ²Growth was followed for up to 96 hr.

TABLE 10 Conversion of D-Xylose to Xylitol via a D-XyluloseIntermediate. Glycerol D-Xylose D-Xylulose Xylitol Strain Host/Plasmid 0hr 24 hr 0 hr 24 hr 0 hr 24 hr 0 hr 24 hr % Con¹ ZUC52 ZUC22/pTrp2280.247 0 0.361 0.356 0 0.000 0 0.001 0.002 ZUC53 ZUC22/pZUC35 0.247 00.361 0.034 0 0.057 0 0.236 65.373 ZUC54 ZUC22/pZUC36 0.247 0 0.3610.031 0 0.053 0 0.245 67.867 % conversion of D-xylose to xylitol in 24hr.

TABLE 11 Utilization of Sugars by ZUC72 Strain/ Time Genotype (hr)D-Glucose D-Xylose L-Arabinose AB707 0 0.071 0.160 0.118 AB707 13 0.0340.179 0.162 AB707 24 0.000 0.048 0.000 ZUC72 0 0.109 0.211 0.167 ZUC7213 0.076 0.197 0.116 ZUC72 24 0.034 0.172 0.018

TABLE 12 Mutations found in xylitol resistant xylose isomerase mutants.Gene Generated Parent Mutations % XTL^(R) WT N/A None <0.5 #1 GeneMorphWT F9L, L213Q, F283Y, K311R, 1 II H420N #3 GeneMorph #1 F9L, Q11K,L213Q, F283Y, 3 II K311R, H420N #8 XL-1 Red #3 F9L, Q11K, S20L, L213Q, 5F283Y, K311R, H420N #9 GeneMorph #8 F9L, Q11K, S20L, L213Q, 10 II F283Y,K311R, H420N, H439Q

1. A recombinant bacterium that expresses proteins comprising: (a)xylose reductase; (b) L-arabitol dehydrogenase or ribitol dehydrogenaseor both; and (c) L-xylulose reductase; wherein the recombinant bacteriumcan produce an end-product of xylitol from substrates comprising:D-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose,D-xylose and other sugars; and wherein no substantial amount ofL-arabitol is produced as an end-product.
 2. The recombinant bacteriumof claim 1, wherein the substrate is a xylan hydrolysate or ahemicellulose hydrolysate.
 3. The recombinant bacterium of claim 1,wherein the bacterium further expresses a ribitol transporter protein.4. The recombinant bacterium of claim 1, wherein the bacterium isEscherichia coli.
 5. The recombinant bacterium of claim 1, wherein thebacterium does not have a ptsG gene or has an inactive ptsG gene.
 6. Therecombinant bacterium of claim 1, wherein the recombinant bacterium isnon-pathogenic.
 7. The recombinant bacterium of claim 1, wherein therecombinant bacterium produces L-arabitol as an intermediate to thexylitol end-product.
 8. The recombinant bacterium of claim 1, whereinthe recombinant bacterium produces L-xylulose as an intermediate to thexylitol end-product.
 9. The recombinant bacterium of claim 1, whereinthe recombinant bacterium comprises one or more recombinant nucleic acidsequences encoding aldose reductase, L-xylose reductase, L-arabitoldehydrogenase, ribitol dehydrogenase, ribitol transporter, andL-xylulose reductase.
 10. The recombinant bacterium of claim 9, whereinthe nucleic acid sequence encoding xylose reductase is a Pichia stipitisnucleic acid sequence.
 11. The recombinant bacterium of claim 9, whereinthe nucleic acid sequence encoding ribitol dehydrogenase is a Klebsiellapneumoniae or Klebsiella aerogenes nucleic acid sequence.
 12. Therecombinant bacterium of claim 9, wherein the nucleic acid sequenceencoding L-xylulose reductase is an Ambrosioyma monospora nucleic acidsequence.
 13. The recombinant bacterium of claim 9, wherein the nucleicacid sequence encoding L-xylose reductase comprises a yafB or yajOnucleic acid sequence from Escherichia coli.
 14. The recombinantbacterium of claim 8, wherein the nucleic acid sequence encodingL-arabitol dehydrogenase is a Trichoderma reesei nucleic acid sequence.15. The recombinant bacterium of claim 9, wherein the nucleic acidsequence encoding L-xylose reductase is a T. reesei nucleic acidsequence.
 16. A method for producing a xylitol end-product comprisingfermenting a substrate comprising D-xylose; or L-arabinose; orL-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars withthe recombinant bacterium of claim
 1. 17. The method of claim 16,wherein L-arabitol is produced as an intermediate to the xylitolend-product.
 18. The method of claim 16, wherein L-xylulose in producedas an intermediate to the xylitol end-product.
 19. The method of claim16, wherein in the method does not require separation of L-arabitol fromthe xylitol end-product.
 20. A method for producing L-xylulosecomprising fermenting a substrate comprising D-xylose; or L-arabinose;or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugarswith the recombinant bacterium of claim 1, and collecting L-xylulosebefore it is converted to xylitol.
 21. A method of producing xylitolfrom a substrate comprising D-xylose; or L-arabinose; or L-arabinose andD-xylose; or L-arabinose, D-xylose and other sugars; comprisingcontacting the substrate with one or more isolated bacteria thatcomprise: (a) xylose reductase activity; (b) L-arabitol dehydrogenaseactivity or ribitol dehydrogenase activity or a combination of both; and(c) L-xylulose reductase activity; wherein the substrate is converted toan end-product of xylitol and wherein substantially no L-arabitol isproduced as an end-product.
 22. The method of claim 21, wherein the oneor more isolated bacteria comprise ribitol transporter activity.
 23. Aprocess for producing xylitol from a substrate comprising D-xylose; orL-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose andother sugars; comprising contacting the substrate with: (a) xylosereductase; (b) L-arabitol dehydrogenase or ribitol dehydrogenase, orboth; and (c) L-xylulose reductase; wherein the substrate is convertedto an end-product of xylitol and wherein substantially no L-arabitol isproduced as an end-product.
 24. The process of claim 23, whereinL-arabitol and L-xylulose are produced as intermediate products.
 25. Theprocess of claim 23, wherein the substrate is further contacted withribitol transporter protein.
 26. An isolated microorganism comprisingxylose specific reductase activity, wherein the xylose specificreductase activity does not convert L-arabinose to L-arabitol.
 27. Theisolated microorganism of claim 26, wherein the microorganism canproduce an end-product of xylitol from a substrate comprising D-xylose;or L-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xyloseand other sugars.
 28. The isolated microorganism of claim 26, whereinthe microorganism produces no substantial amount of L-arabitol as anend-product.
 29. The isolated microorganism of claim 26, wherein thesubstrate is a xylan hydrolysate or hemicellulose hydrolysate.
 30. Theisolated microorganism of claim 26, wherein the microorganism is E.coli.
 31. The isolated microorganism of claim 26, wherein themicroorganism does not have a ptsG gene or has an inactive ptsG gene.32. The isolated microorganism of claim 26, wherein the recombinantmicroorganism is non-pathogenic.
 33. The isolated microorganism of claim26, wherein the microorganism is a bacteria, fungus or yeast.
 34. Theisolated microorganism of claim 26, wherein the xylose specificreductase is encoded by a nucleic acid comprising SEQ ID NO:43.
 35. Amethod for producing xylitol comprising fermenting a substratecomprising D-xylose; or L-arabinose; or L-arabinose and D-xylose; orL-arabinose, D-xylose and other sugars; with the isolated microorganismof claim
 26. 36. The method of claim 35, wherein the method does notrequire separation of L-arabitol from xylitol.
 37. A purified xylosespecific reductase comprising SEQ ID NO:43.
 38. A purified P. stipitisxylose reductase comprising a Ser233Pro mutation and a Phe286Leumutation.
 39. A process for producing xylitol comprising contacting asubstrate comprising D-xylose; or L-arabinose; or L-arabinose andD-xylose; or L-arabinose, D-xylose and other sugars; with axylose-specific reductase.
 40. The process of claim 39, wherein thexylose-specific reductase comprises SEQ ID NO:43.
 41. A recombinant E.coli that comprises a nucleic acid sequence encoding xylitoldehydrogenase, wherein the E. coli produces substantially no xyloseisomerase.
 42. The recombinant E. coli of claim 41, wherein the E. colidoes not have a ptsG gene or has an inactive ptsG gene.
 43. A method ofscreening for xylose reductase activity comprising: transforming therecombinant E. coli of claim 41 with a nucleic acid molecule encoding aputative xylose reductase to produce a transformant; and adding thetransformant to D-xylose minimal media, wherein, if the transformantcomprises an expressed nucleic acid encoding a xylose reductase thetransformant will grow in the D-xylose minimal media.
 44. A recombinantE. coli comprising L-xylulose kinase activity, L-xylulose 5-phosphateepimerase activity, and L-ribulose 5-phosphate 4-epimerase activity. 45.The recombinant E. coli strain of claim 44, wherein the strain is strainK12.
 46. A recombinant E. coli strain comprising a deleted or inactiveyiaJ gene.
 47. The recombinant E. coli strain of claim 46, wherein thestrain is strain K12.
 48. A method of screening for L-arabitoldehydrogenase activity or ribitol dehydrogenase activity comprising:transforming the recombinant E. coli of claim 44 with a nucleic acidmolecule encoding a putative L-arabinitol dehydrogenase or ribitoldehydrogenase to produce a transformant, and adding the transformant toL-arabinitol media, wherein if the transformant comprises an expressednucleic acid encoding a L-arabinitol dehydrogenase or ribitoldehydrogenase, the transformant will grow in the L-arabinitol media. 49.A method of screening for L-arabitol dehydrogenase activity or ribitoldehydrogenase activity comprising: transforming the recombinant E. coliof claim 46 with a nucleic acid molecule encoding a putativeL-arabinitol dehydrogenase or ribitol dehydrogenase to produce atransformant, and adding the transformant to L-arabinitol media, whereinif the transformant comprises an expressed nucleic acid encoding aL-arabinitol dehydrogenase or ribitol dehydrogenase, the transformantwill grow in the L-arabinitol media.
 50. A purified xylose reductasethat is active at 37° C.
 51. The purified xylose reductase of claim 50,wherein the xylose reductase retains 90% or more of its activity at 37°C. when compared to its activity at 30° C.
 52. The purified xylosereductase of claim 50, wherein the xylose reductase comprises an aminoacid sequence of SEQ ID NO:44.
 53. The purified xylose reductase ofclaim 50, wherein the xylose reductase comprises a C. tenuis xylosereductase that comprises a Gly32Ser mutation and a Asn138Asp mutation.54. A method for screening for bacteria that cannot utilize L-arabinosecomprising: transforming bacteria that do not have xylose isomeraseactivity or an araBAD operon and that have xylitol dehydrogenaseactivity and L-ribulokinase activity, with a nucleic acid encoding axylose reductase, wherein if the xylose reductase is a xylose-specificreductase the transformed bacteria cannot utilize L-arabinose and willgrow on media comprising L-arabinose and D-xylose, and wherein if thexylose reductase is not a xylose-specific reductase, the transformedbacteria can utilize L-arabinose and will not grow on media comprisingL-arabinose and D-xylose.
 55. An isolated microorganism comprising arecombinant operon comprising a nucleic acid encoding a xylitoldehydrogenase and a nucleic acid encoding a xylose isomerase.
 56. Amethod of converting D-xylose to xylitol comprising fermenting asubstrate comprising D-xylose with the isolated microorganism of claim55.
 57. The method of claim 54, wherein D-xylulose is produced as anintermediate to the xylitol.
 58. The method of claim 54, wherein greaterthan 50% of the D-xylose is converted to xylitol.
 59. The method ofclaim 54, wherein the microorganism is a bacteria, fungus, or yeast. 60.The method of claim 54, wherein the microorganism is E. coli.
 61. Themethod of claim 54, wherein the microorganism does not have a ptsG geneor has an inactive ptsG gene.
 62. A purified E. coli xylose isomerase(xylA) wherein the amino acid sequence comprises the followingmutations: (a) F9L, L213Q, F283Y, K311R, H420N; (b) F9L, Q11K, L213Q,F283Y, K311R, H420N; (c) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N; or(d) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N, H439Q.
 63. An isolatednucleic acid molecule encoding the E. coli xylose isomerase of claim 62.64. A recombinant microorganism comprising a mutated E. coli xyloseisomerase coding sequence, wherein the microorganism can grow in thepresence of about 1% or more of xylitol.
 65. The recombinantmicroorganism of claim 64, wherein the mutated E. coli xylose isomerasecoding sequence encodes the following mutations: (a) F9L, L213Q, F283Y,K311R, H420N; (b) F9L, Q11K, L213Q, F283Y, K311R, H420N; (c) F9L, Q11K,S20L, L213Q, F283Y, K311R, H420N; or (d) F9L, Q11K, S20L, L213Q, F283Y,K311R, H420N, H439Q.